Modulation of the NF-κB Pathway by Bordetella pertussis Filamentous Hemagglutinin

Background Filamentous hemagglutinin (FHA) is a cell-associated and secreted adhesin produced by Bordetella pertussis with pro-apoptotic and pro-inflammatory activity in host cells. Given the importance of the NF-κB transcription factor family in these host cell responses, we examined the effect of FHA on NF-κB activation in macrophages and bronchial epithelial cells, both of which are relevant cell types during natural infection. Methodology/Principal Findings Exposure to FHA of primary human monocytes and transformed U-937 macrophages, but not BEAS-2B epithelial cells, resulted in early activation of the NF-κB pathway, as manifested by the degradation of cytosolic IκBα, by NF-κB DNA binding, and by the subsequent secretion of NF-κB-regulated inflammatory cytokines. However, exposure of macrophages and human monocytes to FHA for two hours or more resulted in the accumulation of cytosolic IκBα, and the failure of TNF-α to activate NF-κB. Proteasome activity was attenuated following exposure of cells to FHA for 2 hours, as was the nuclear translocation of RelA in BEAS-2B cells. Conclusions These results reveal a complex temporal dynamic, and suggest that despite short term effects to the contrary, longer exposures of host cells to this secreted adhesin may block NF-κB activation, and perhaps lead to a compromised immune response to this bacterial pathogen

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

Lipopolysaccharide‐induced inflammation does not alter muscle spindle afferent mechanosensation or sensory integration in the spinal cord of adult mice

Inflammation is known to alter nervous system function, but its effect on muscle spindle afferent mechanosensation and sensory integration in the spinal cord has not been well studied. We tested the hypothesis that systemic inflammation induced by an intraperitoneal injection of the endotoxin lipopolysaccharide (LPS; 7.5 × 105 endotoxin units/kg 18 h before experiment) would alter muscle spindle afferent mechanosensation and spinal cord excitability to Group Ia input in male and female adult C57Bl/6 mice. LPS injection caused a systemic immune response, evidenced by decreased white blood cell, monocyte, and lymphocyte concentrations in the blood, increased blood granulocyte concentration, and body weight loss. The immune response in both sexes was qualitatively similar. We used an in vitro muscle‐nerve preparation to assay muscle spindle afferent response to stretch and vibration. LPS injection did not significantly change the response to stretch or vibration, with the exception of small decreases in the ability to entrain to high‐frequency vibration in male mice. Similarly, LPS injection did not alter spinal cord excitability to Group Ia muscle spindle afferent input as measured by the Hoffman\u27s reflex test in anesthetized mice (100 mg/kg ketamine, 10 mg/kg xylazine). Specifically, there were no changes in M or H wave latencies nor in the percentage of motor neurons excited by electrical afferent stimulation (Hmax/Mmax). Overall, we found no major alterations in muscle proprioceptor function or sensory integration following exposure to LPS at a dose and time course that causes changes in nociceptor function and central processing

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

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

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

Functional effects of α4 integrins on migration and adhesion of blood leukocytes from <i>B. pertussis</i> and <i>B. parapertussis</i> infections <i>in vitro</i>.

<p>(<b>A</b>) Migration of blood leukocytes from <i>B. pertussis</i> and <i>B. parapertussis</i> infections at 5 days p.i. through vascular cell adhesion molecule 1 (VCAM-1)-coated or BSA-coated transwells, in response to stromal cell-derived factor-1 alpha (SDF-1α) or control medium. The number of leukocytes derived from <i>B. parapertussis</i> cells through VCAM-1 towards SDF-1α is 100% by definition. Error bars represent the SEM of three independent experiments. In each experiment, four mice were used per treatment group. A two-tailed student's <i>t</i>-test was performed; *: p<0.05. (<b>B</b>) Adhesion of blood leukocytes at 5 days p.i. to 96-well plates pre-coated with VCAM-1 (10 µg/ml) or BSA, from <i>B. pertussis</i> and <i>B. parapertussis</i> infections. After 30 minutes, adherents were stained with crystal violet. Values represent mean A₅₉₅ of the solubilized dyes. Error bars represent the SEM of six independent observations. A two-tailed student's <i>t</i>-test was performed; *: p<0.05 between <i>B. pertussis</i> and <i>B. parapertussis</i> infection groups.</p

Antibodies used to stain T cells.

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