<i>In Vitro</i> Inflammation Inhibition Model Based on Semi-Continuous Toll-Like Receptor Biosensing

<div><p>A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an <i>in vivo</i>-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., <i>Pseudomonas aeruginosa</i>) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-κB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests.</p></div

Application of the semi-continuous analytical approach for TLR regulation to test the anti-inflammatory substance effect.

<p>Optimal conditions for the TLR regulation scheme were determined (A) and TLR expression was observed in response to two cyclic repeated bacterial stimulations (B, No treatment). Sodium salicylate was the positive inhibitor (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g002" target="_blank">Figure 2</a>) and an effective dose (50 mM) was used either by co-incubation with the stimulus agent (Co-addition) or by pre-incubation (Pre-incubation). Both treatments revealed inhibition of the TLR response to serial stimulations, suggesting a model for anti-inflammatory substance screening. Each measurement was repeated twice under identical conditions and comparisons to the control (No treatment) were marked as stated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g002" target="_blank">Figure 2</a> legend.</p

Mechanism of inflammatory response via NF-κB activation against bacterial invasion.

<p>Inflammation proceeds mainly via two different routes, which are initiated by TLRs-PAMPs binding (Infection process) or bradykinin-BR interactions (Inflammation process). The former invokes inflammation by producing cytokines as a result of the response. NF-κB activation also increases the densities of other mediators such as TLRs and BRs.</p

Simulation of anti-inflammatory agent screening based on standard controls in the repetitive inhibition response curve.

<p>According to the testing scheme (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g003" target="_blank">Figure 3A</a>), the stimulation-and-restoration cycle was repetitively maintained three times without chemical treatment (no inhibition) or separately with sodium salicylate treatment (positive inhibition). When two different substances were employed in the inhibition testing, CAPE (90 µM) showed positive anti-inflammatory response (A) when compared with the two control curves, whereas acetaminophen (10 mM) revealed a negative inhibition response (B). All experiments were carried out in duplicate and comparisons to the control (No inhibition) were marked as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105212#pone-0105212-g002" target="_blank">Figure 2</a> legend.</p

Simulation of the drug effect persistency testing using sodium salicylate.

<p>Pre-incubation with sodium salicylate during each cycle caused consistent anti-inflammatory responses to the three repeated bacterial stimulations (“1,2,3-inhibition” as a positive control) compared to that without inhibition (Non-inhibition as negative control). However, when the treatment was skipped at either the second (“1,3-inhibition”) or third cycle (“1,2-inhibition”), the inhibitory effect was not shown at the corresponding stage. This may indicate that drug efficacy persisted for 1 day. Each measurement was repeated twice under the same conditions and the significance was indicated.</p

Toll-Like Receptor-Based Immuno-Analysis of Pathogenic Microorganisms

In this study, a novel mammalian cell receptor-based immuno-analytical method was developed for the detection of food-poisoning microorganisms by employing toll-like receptors (TLRs) as sensing elements. Upon infection with bacterium, the host cells respond by expressing TLRs, particularly TLR1, TLR2, and TLR4, on the outer membrane surfaces. To demonstrate the potential of using this method for detection of foodborne bacteria, we initially selected two model sensing systems, expression of TLR1 on a cell line, A549, for <i>Escherichia coli</i> and TLR2 on a cell line, RAW264.7, for <i>Shigella sonnei</i> (<i>S. sonnei</i>). Each TLR was detected using antibodies specific to the respective marker. We also found that the addition of immunoassay for the pathogen captured by the TLRs on the mammalian cells significantly enhanced the detection capability. A dual-analytical system for <i>S. sonnei</i> was constructed and successfully detected an extremely low number (about 3.2 CFU per well) of the pathogenic bacterium 5.1 h after infection. This detection time was 2.5 h earlier than the time required for detection using the conventional immunoassay. To endow the specificity of detection, the target bacterium was immuno-magnetically concentrated by a factor of 50 prior to infection. This further shortened the response to approximately 3.4 h, which was less than half of the time needed when the conventional method was used. Such enhanced performance could basically result from synergistic effects of bacterial dose increase and subsequent autocrine signaling on TLRs’ up-regulation upon infection with live bacterium. This TLR-based immuno-sensing approach may also be expanded to monitor infection of the body, provided scanning of the signal is feasible