Differential gene expression in TECs exposed to LAMPs in the presence of signaling inhibitors.

<p>Epithelial cells were exposed to LAMPs isolated from R_low or R_high in the presence or absence of signaling inhibitors for 6 hours. Samples were normalized to the housekeeping gene GAPDH and un-exposed TECs served as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. <b>A</b>. IL-12p40. <b>B</b>. IL-1β. <b>C</b>. IL-8. <b>D</b>. IL-6. <b>E.</b> CCL-20. <b>F</b>. NOS-2.</p

Primary chicken tracheal epithelial cell culture (TEC).

<p>Primary chicken tracheal epithelial cells were isolated and cultured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112796#s2" target="_blank">Methods</a> section. <b>1A:</b> Primary chicken tracheal epithelial cells at 100X magnification. <b>1B:</b> Confirmation of tracheal epithelial cell identity both <i>in vitro</i> and freshly isolated (<i>ex vivo</i>) from tracheae after <i>ex-vivo</i> exposure: PCR amplified epithelial cell specific genes from cDNA in agarose gel, compared to chicken embryonic fibroblast (DF-1) cells. <b>1C:</b> Tracheal epithelial cells stained for E-cadherin and Vimentin at (400X magnification). Left panel shows TECs at different filter setting Blue (DAPI) for nuclear staining, Green (FITC) for Vimentin and Red (AlexaFluor 546) for E-cadherin, right panel shows merged picture for all filters. <b>1D:</b> DF-1 fibroblast cells stained for E-cadherin and Vimentin at 400X magnification. Left panel shows DF-1 cells at different filter setting; Blue (DAPI) for nuclear staining, Green (FITC) for Vimentin and Red (AlexaFluor 546) for E-cadherin; right panel shows merged picture for all filters.</p

<i>Mycoplasma gallisepticum</i> Lipid Associated Membrane Proteins Up-regulate Inflammatory Genes in Chicken Tracheal Epithelial Cells via TLR-2 Ligation through an NF-κB Dependent Pathway

<div><p><i>Mycoplasma gallisepticum</i>-mediated respiratory inflammation in chickens is associated with accumulation of leukocytes in the tracheal submucosa. However the molecular mechanisms underpinning these changes have not been well described. We hypothesized that the initial inflammatory events are initiated upon ligation of mycoplasma lipid associated membrane proteins (LAMP) to TLRs expressed on chicken tracheal epithelial cells (TEC). To test this hypothesis, live bacteria or LAMPs isolated from a virulent (R_low) or a non-virulent (R_high) strain were incubated with primary TECs or chicken tracheae <i>ex vivo</i>. Microarray analysis identified up-regulation of several inflammatory and chemokine genes in TECs as early as 1.5 hours post-exposure. Kinetic analysis using RT-qPCR identified the peak of expression for most genes to be at either 1.5 or 6 hours. <i>Ex-vivo</i> exposure also showed up-regulation of inflammatory genes in epithelial cells by 1.5 hours. Among the commonly up-regulated genes were IL-1β, IL-6, IL-8, IL-12p40, CCL-20, and NOS-2, all of which are important immune-modulators and/or chemo-attractants of leukocytes. While these inflammatory genes were up-regulated in all four treatment groups, R_low exposed epithelial cells both <i>in vitro</i> and <i>ex vivo</i> showed the most dramatic up-regulation, inducing over 100 unique genes by 5-fold or more in TECs. Upon addition of a TLR-2 inhibitor, LAMP-mediated gene expression of IL-1β and CCL-20 was reduced by almost 5-fold while expression of IL-12p40, IL-6, IL-8 and NOS-2 mRNA was reduced by about 2–3 fold. Conversely, an NF-κB inhibitor abrogated the response entirely for all six genes. miRNA-146a, a negative regulator of TLR-2 signaling, was up-regulated in TECs in response to either R_low or R_high exposure. Taken together we conclude that LAMPs isolated from both R_high and R_low induced rapid, TLR-2 dependent but transient up-regulation of inflammatory genes in primary TECs through an NF-κB dependent pathway.</p></div

Differential gene expression in TECs post-exposure.

<p>mRNA fold difference in TECs exposed to R_low, R_low LAMP, R_high or R_high LAMP at 1.5, 6 and 24 hours respectively. Samples normalized to housekeeping gene GAPDH and un-exposed TECs as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. <b>A:</b> IL-12p40 mRNA. <b>B:</b> IL-8 mRNA. <b>C:</b> IL-6 mRNA. <b>D:</b> CCL-20 mRNA. <b>E:</b> NOS-2 mRNA. <b>F:</b> IL-1β mRNA.</p

RT-qPCR analysis of miRNA and IL-10 differential expression in TECs.

<p>Epithelial cells were exposed to R_low, R_low LAMP, R_high or R_high LAMP at 1.5, 6 and 24 hours respectively. Samples were normalized to housekeeping gene GAPDH and un-exposed TECs as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. <b>A:</b> mRNA fold difference of IL-10 in TECs at all three time points post exposure. <b>B:</b> mRNA fold difference of miRNA-146a in TECs at all three time points post exposure.</p

Genes of significant interest from microarray analysis.

<p>Representative list of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112796#pone.0112796.s005" target="_blank">Table S1</a>: mRNA fold changes ≥2 (p-value ≤0.05) in TECs exposed to R_low, R_high, R_low LAMP and R_high LAMP compared to control.</p><p>Genes of significant interest from microarray analysis.</p

Differential gene expression in tracheal epithelial cells after <i>ex-vivo</i> exposure to LAMPs.

<p>Comparison of mRNA fold difference in tracheal epithelial cells from tracheal explant exposed to R_low, R_low LAMP, R_high or R_high LAMP at 1.5 and 6 hours respectively. Samples normalized to housekeeping gene GAPDH and un-exposed tracheae as control. n = 6 for all experiments. Results are denoted as fold change ± SEM with all control values set at 1. Significant differences denoted as * = P<0.05, ** = P<0.01, *** = P<0.001. <b>A:</b> mRNA fold difference of all genes at 1.5 hours. <b>B:</b> mRNA fold difference of all genes at 6 hours.</p

Distribution of differentially regulated genes in TECs.

<p>Differentially regulated genes (≥5 fold) in tracheal epithelial cell after exposure to live R_low, R_high or LAMPs isolated from either strain 1.5 hours after exposure. The star (*) in the figure represent commonly up-regulated genes upon all four exposures, from which six follow up genes were chosen. n = 8 (4 biological replicates x2 dye swap technical replicates) for all microarray experiments.</p

Gene specific primers for RT-qPCR.

<p>Gene specific primers for RT-qPCR.</p

A Microarray Biosensor for Multiplexed Detection of Microbes Using Grating-Coupled Surface Plasmon Resonance Imaging

Grating-coupled surface plasmon resonance imaging (GCSPRI) utilizes an optical diffraction grating embossed on a gold-coated sensor chip to couple collimated incident light into surface plasmons. The angle at which this coupling occurs is sensitive to the capture of analyte at the chip surface. This approach permits the use of disposable biosensor chips that can be mass-produced at low cost and spotted in microarray format to greatly increase multiplexing capabilities. The current GCSPRI instrument has the capacity to simultaneously measure binding at over 1000 unique, discrete regions of interest (ROIs) by utilizing a compact microarray of antibodies or other specific capture molecules immobilized on the sensor chip. In this report, we describe the use of GCSPRI to directly detect multiple analytes over a large dynamic range, including soluble protein toxins, bacterial cells, and viruses, in near real-time. GCSPRI was used to detect a variety of agents that would be useful for diagnostic and environmental sensing purposes, including macromolecular antigens, a nontoxic form of <i>Pseudomonas aeruginosa</i> exotoxin A (ntPE), <i>Bacillus globigii</i>, <i>Mycoplasma hyopneumoniae</i>, <i>Listeria monocytogenes</i>, <i>Escherichia coli</i>, and M13 bacteriophage. These studies indicate that GCSPRI can be used to simultaneously assess the presence of toxins and pathogens, as well as quantify specific antibodies to environmental agents, in a rapid, label-free, and highly multiplexed assay requiring nanoliter amounts of capture reagents