The Combination of IFN ß and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9.

Interferon (IFN) ß and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNß is reprogrammed by crosstalk with TNF. IFNß mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFNß and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNß and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFNß and TNF.

Interleukin (IL)-6 Inhibits IL-27- and IL-30-Mediated Inflammatory Responses in Human Monocytes.

Interleukin (IL)-30, the IL-27p28 subunit of the heterodimeric cytokine IL-27, acts as an antagonist of IL-27 and IL-6 signaling in murine cells via glycoprotein 130 (gp130) receptor and additional binding partners. Thus far, functions of IL-30 have not been fully elucidated in human cells. We demonstrate that like IL-27, IL-30 upregulated TLR4 expression to enhance lipopolysaccharide-induced TNF-α production in human monocytes; however, these IL-30-mediated activities did not reach the same levels of cytokine induction compared to IL-27. Interestingly, IL-30- and IL-27-mediated interferon-γ-induced protein 10 (IP-10) production required WSX-1 engagement and signal transducer and activator of transcription (STAT) 3 phosphorylation; furthermore, IL-30 induced STAT phosphorylation after 16 h, whereas IL-27 induced STAT phosphorylation within 30 min. This prompted us to examine if a secondary mediator was required for IL-30-induced pro-inflammatory functions, and hence we examined IL-6-related molecules. Combined with inhibition of soluble IL-6 receptor α (sIL-6Rα) and data showing that IL-6 inhibited IL-30/IL-27-induced IP-10 expression, we demonstrate a role for sIL-6Rα and gp130 in IL-30-mediated activity in human cells.

DUSP1 regulates apoptosis and cell migration, but not the JIP1-protected cytokine response, during Respiratory Syncytial Virus and Sendai Virus infection.

The host antiviral response involves the induction of interferons and proinflammatory cytokines, but also the activation of cell death pathways, including apoptosis, to limit viral replication and spreading. This host defense is strictly regulated to eliminate the infection while limiting tissue damage that is associated with virus pathogenesis. Post-translational modifications, most notably phosphorylation, are key regulators of the antiviral defense implying an important role of protein phosphatases. Here, we investigated the role of the dual-specificity phosphatase 1 (DUSP1) in the host defense against human respiratory syncytial virus (RSV), a pathogenic virus of the Pneumoviridae family, and Sendai virus (SeV), a model virus being developed as a vector for anti-RSV vaccine. We found that DUSP1 is upregulated before being subjected to proteasomal degradation. DUSP1 does not inhibit the antiviral response, but negatively regulates virus-induced JNK/p38 MAPK phosphorylation. Interaction with the JNK-interacting protein 1 scaffold protein prevents dephosphorylation of JNK by DUSP1, likely explaining that AP-1 activation and downstream cytokine production are protected from DUSP1 inhibition. Importantly, DUSP1 promotes SeV-induced apoptosis and suppresses cell migration in RSV-infected cells. Collectively, our data unveils a previously unrecognized selective role of DUSP1 in the regulation of tissue damage and repair during infections by RSV and SeV.

A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3.

The IRF3 transcription factor is critical for the first line of defense against pathogens mainly through interferon ß and antiviral gene expression. A detailed analysis of IRF3 activation is essential to understand how pathogens induce or evade the innate antiviral response. Distinct activated forms of IRF3 can be distinguished based on their phosphorylation and monomer vs dimer status. In vivo discrimination between the different activated species of IRF3 can be achieved through the separation of IRF3 phosphorylated forms based on their mobility shifts on SDS-PAGE. Additionally, the levels of IRF3 monomer and dimer can be monitored using non-denaturing electrophoresis. Here, we detail a procedure to reach the highest resolution to gain the most information regarding IRF3 activation status. This is achieved through the combination of a high resolution SDS-PAGE and a native-PAGE coupled to immunoblots using multiple total and phosphospecific antibodies. This experimental strategy constitutes an affordable and sensitive approach to acquire all the necessary information for a complete analysis of the phosphorylation-mediated activation of IRF3.

Lung epithelial NOX/DUOX and respiratory virus infections.

Determining the role of NADPH oxidases in the context of virus infection is an emerging area of research and our knowledge is still sparse. The expression of various isoforms of NOX/DUOX (NADPH oxidase/dual oxidase) in the epithelial cells (ECs) lining the respiratory tract renders them primary sites from which to orchestrate the host defence against respiratory viruses. Accumulating evidence reveals distinct facets of the involvement of NOX/DUOX in host antiviral and pro-inflammatory responses and in the control of the epithelial barrier integrity, with individual isoforms mediating co-operative, but surprisingly also opposing, functions. Although in vivo studies in mice are in line with some of these observations, a complete understanding of the specific functions of epithelial NOX/DUOX awaits lung epithelial-specific conditional knockout mice. The goal of the present review is to summarize our current knowledge of the role of individual NOX/DUOX isoforms expressed in the lung epithelium in the context of respiratory virus infections so as to highlight potential opportunities for therapeutic intervention.

Label-free in vitro visualization and characterization of caveolar bulbs during stimulated re-epithelialization.

Tip-enhanced Raman scattering (TERS) was paired with real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) to characterize lipid aggregates during stimulated re-epithelialization using an in vitro wound healing model. In this study, lipid fluctuations in the plasma membrane of epidermal keratinocytes were studied at multiple time points post-wounding. TERS measurements for the first time were also combined with sample analysis after initial wounding and 24 h of wound healing. This enabled simultaneous visualization and characterization of caveolar bulb distribution during wound healing stages, providing noninvasive insight into their associated lipid structure and coating protein, caveolin, in the nanometer range. The combination of Raman spectroscopy and scanning probe microscopy in TERS gives access to topographic and chemical structure information in a single experiment. It is the intrinsic specificity and sensitivity of TERS that enable this discrete detection of cell surface components on the nanometer scale. In contrast with competing biochemical methods, the applied technique does not interfere with the cellular composition, enabling lipid structure analysis without digestion or detergents, and displayed great potential for future biological in vivo studies.

Applications of modern micro-Raman spectroscopy for cell analyses.

Raman spectroscopy assesses the chemical composition of a sample by exploiting the inherent and unique vibrational characteristics of chemical bonds. Initial applications of Raman were identified in the industrial and chemical sectors, providing a rapid non-invasive method to identify sample components or perform quality control assessments. Applications have since increased and sample sizes decreased, leading to the onset of micro-Raman spectroscopy. Coupling with microscopy enabled label-free sample analysis and the unveiling of total chemical composition. Latter adaptations of Raman have advanced into biomedical diagnostics and research. Alongside technical developments in filter systems and detectors, spectral peak intensities and improved signal-to-noise ratios have facilitated target molecule measurement within a variety of samples. Quantitative sample analysis applications of Raman have contributed to its increasing popularity. Through these exceptional capabilities, potential Raman spectroscopy utility in biomedical research applications has expanded, exemplifying why there is continued interest in this highly sensitive and often under-used technique.

Separation of CARS image contributions with a Gaussian mixture model.

Coherent anti-Stokes Raman scattering (CARS) gained a lot of importance in chemical imaging. This is due to the fast image acquisition time, the high spatial resolution, the non-invasiveness, and the molecular sensitivity of this method. By using the single-line CARS in contrast to the multiplex CARS, different signal contributions stemming from resonant and non-resonant light-matter interactions are indistinguishable. Here a numerical method is presented in order to extract more information from univariate CARS images: vibrational composition, morphological information, and contributions from index-of-refraction steps can be separated from single-line CARS images. The image processing algorithm is based on the physical properties of CARS process as reflected in the shape of the intensity histogram of univariate CARS images. Because of this the comparability of individual CARS images recorded with different experimental parameters is achieved. The latter is important for a quantitative evaluation of CARS images.

Micro-Raman detection of nuclear membrane lipid fluctuations in senescent epithelial breast cancer cells.

Originally identified in cultured cells, oncogenic cellular senescence is a growth-arrest mechanism which may inhibit tumor development by limiting the ability of cells to divide. However, literature shows that these cells secrete tumor-inducing and tumor-suppressing proteins leading to poor prognosis. Understanding the progression of oncogenic cellular senescence and associated mechanisms provides important implications for improving tumorigenesis therapeutic treatments. Micro-Raman spectroscopic imaging has grown in popularity as an imaging technique compared to the standard imaging predecessors and can be attributed to its numerous benefits such as no sample perturbation and the provision of direct chemical information. Through the use of label-free micro-Raman spectroscopy, control and senescent cells were noninvasively imaged. Resulting spectral images were processed using chemometric techniques, and average nuclei spectra from each sample set were compared. In turn, changes in the -cis and -trans unsaturated lipid isomer content were found to differ among proliferating and senescent cells. This may lead to increased nuclear fluidity and may contribute to the inability of senescent cells to complete the cell cycle. In the tumor environment, this detected increase in nuclear envelope fluidity could lead to downstream gene expression modifications and increased nucleo-cytoplasmic RNA translocation. Understanding nuclear envelope fluidity could provide insight into secretory profiles of senescent cells and their role in carcinogenesis, meriting further investigation into novel therapeutic technique development for oncogenic cellular senescence.

Impact of fixation on in vitro cell culture lines monitored with Raman spectroscopy.

Raman spectroscopy provides chemical-rich information about the composition of analytes and is a powerful tool for biological studies. With the ability to investigate specific cellular components or image whole cells, compatible methods of sample preservation must be implemented for accurate spectra to be collected. Unfortunately, the effects of many commonly used sample preservation methods have not been explored with cultured cells. In this study, two human cell lineages of varying phenotypes were used to investigate the effects of sample preservation methods. Cells were cultured directly onto quartz substrates and either formalin-fixed, desiccated or air dried. The results indicate that the methodology applied to cell cultures for Raman analysis significantly influences the quality and reproducibility of the resulting spectral data. Formalin fixation was not found to be as universally efficient as anticipated for a commonly used fixative. This was due largely to the inconsistency in sample preservation between cell lines and loss of signal intensity. Sample air-drying was found to be largely inconsistent in terms of spectral reproducibility. Our study shows that sample desiccation displayed good spectral reproducibility and resulted in a good signal-to-noise ratio. Lipid and protein content in both activated and inactivated cells were maintained and provided a more controlled method compared with air-drying, revealing that the speed of drying is important for sample preservation.