The innate immune system processes pathogen-induced signals into cell fate decisions. How information is turned to decision remains unknown. By combining stochastic mathematical modelling and experimentation, we demonstrate that feedback interactions between the IRF3, NF-κB and STAT pathways lead to switch-like responses to a viral analogue, poly(I:C), in contrast to pulse-like responses to bacterial LPS. Poly(I:C) activates both IRF3 and NF-κB, a requirement for induction of IFNβ expression. Autocrine IFNβ initiates a JAK/STAT-mediated positive-feedback stabilising nuclear IRF3 and NF-κB in first responder cells. Paracrine IFNβ, in turn, sensitises second responder cells through a JAK/STAT-mediated positive feedforward pathway that upregulates the positive-feedback components: RIG-I, PKR and OAS1A. In these sensitised cells, the ‘live-or-die’ decision phase following poly(I:C) exposure is shorter—they rapidly produce antiviral responses and commit to apoptosis. The interlinked positive feedback and feedforward signalling is key for coordinating cell fate decisions in cellular populations restricting pathogen spread.

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1–2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.

This paper presents the development and comparison of label-free electrochemical immunosensors based on screen-printed gold and glassy carbon (GC) disc electrodes for efficient and rapid detection of respiratory syncytial virus (RSV). Briefly, the antibody specific to the F protein of RSV was successfully immobilized on modified electrodes. Antibody coupling on the Au surface was conducted via 4-aminothiophenol (4-ATP) and glutaraldehyde (GA). The GC surface was modified with poly-L-lysine (PLL) for direct anti-RSV conjugation after EDC/NHS (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-Hydroxysuccinimide) activation. Electrochemical characterizations of the immunosensors were carried out by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). GC-based immunosensors show a dynamic range of antigen detection from 1.0 × 10⁵ PFU/mL to 1.5×10⁷ PFU/mL, more than 1.0 × 10⁵ PFU/mL to 1.0 × 10⁷ PFU/mL for the Au-based sensor. However, the GC platform is less sensitive and shows a higher detection limit (LOD) for RSV. The limit of detection of the Au immunosensor is 1.1 × 10³ PFU/mL, three orders of magnitude lower than 2.85 × 10⁶ PFU/mL for GC. Thus, the Au-based immunosensor has better analytical performance for virus detection than a carbon-based platform due to high sensitivity and very low RSV detection, obtained with good reproducibility.

Influenza A virus (IAV) and respiratory syncytial virus (RSV) are common recurrent respiratory infectants that show a relatively high coincidence. We demonstrated that preinfection with RSV partitions the cell population into a subpopulation susceptible to subsequent infection with IAV and an IAV-proof subpopulation.

The NF-κB pathway is known to transmit merely 1 bit of information about stimulus level. We combined experimentation with mathematical modeling to elucidate how information about TNF concentration is turned into a binary decision. Using Kolmogorov-Smirnov distance, we quantified the cell’s ability to discern 8 TNF concentrations at each step of the NF-κB pathway, to find that input discernibility decreases as signal propagates along the pathway. Discernibility of low TNF concentrations is restricted by noise at the TNF receptor level, whereas discernibility of high TNF concentrations it is restricted by saturation/depletion of downstream signaling components. Consequently, signal discernibility is highest between 0.03 and 1 ng/ml TNF. Simultaneous exposure to TNF or LPS and a translation inhibitor, cycloheximide, leads to prolonged NF-κB activation and a marked increase of transcript levels of NF-κB inhibitors, IκBα and A20. The impact of cycloheximide becomes apparent after the first peak of nuclear NF-κB translocation, meaning that the NF-κB network not only relays 1 bit of information to coordinate the all-or-nothing expression of early genes, but also over a longer time course integrates information about other stimuli. The NF-κB system should be thus perceived as a feedback-controlled decision-making module rather than a simple information transmission channel.