Shedding light on the molecular and regulatory mechanisms of TLR4 signaling in endothelial cells under physiological and inflamed conditions.

Toll-like receptor 4 (TLR4) are part of the innate immune system. They are capable of recognizing pathogen-associated molecular patterns (PAMPS) of microbes, and damage-associated molecular patterns (DAMPs) of damaged tissues. Activation of TLR4 initiates downstream signaling pathways that trigger the secretion of cytokines, type I interferons, and other pro-inflammatory mediators that are necessary for an immediate immune response. However, the systemic release of pro-inflammatory proteins is a powerful driver of acute and chronic inflammatory responses. Over the past decades, immense progress has been made in clarifying the molecular and regulatory mechanisms of TLR4 signaling in inflammation. However, the most common strategies used to study TLR4 signaling rely on genetic manipulation of the TLR4 or the treatment with agonists such as lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, which are often associated with the generation of irreversible phenotypes in the target cells or unintended cytotoxicity and signaling crosstalk due to off-target or pleiotropic effects. Here, optogenetics offers an alternative strategy to control and monitor cellular signaling in an unprecedented spatiotemporally precise, dose-dependent, and non-invasive manner. This review provides an overview of the structure, function and signaling pathways of the TLR4 and its fundamental role in endothelial cells under physiological and inflammatory conditions, as well as the advances in TLR4 modulation strategies.

An Update on Toll-like Receptor 2, Its Function and Dimerization in Pro- and Anti-Inflammatory Processes.

While a certain level of inflammation is critical for humans to survive infection and injury, a prolonged inflammatory response can have fatal consequences. Pattern recognition Toll-like receptors (TLRs) are key players in the initiation of an inflammatory process. TLR2 is one of the most studied pattern recognition receptors (PRRs) and is known to form heterodimers with either TLR1, TLR4, TLR6, and TLR10, allowing it to recognize a wide range of pathogens. Although a large number of studies have been conducted over the past decades, there are still many unanswered questions regarding TLR2 mechanisms in health and disease. In this review, we provide an up-to-date overview of TLR2, including its homo- and heterodimers. Furthermore, we will discuss the pro- and anti-inflammatory properties of TLR2 and recent findings in prominent TLR2-associated infectious and neurodegenerative diseases.

Beyond Pattern Recognition: TLR2 Promotes Chemotaxis, Cell Adhesion, and Migration in THP-1 Cells.

The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.

Comparative and Temporal Characterization of LPS and Blue-Light-Induced TLR4 Signal Transduction and Gene Expression in Optogenetically Manipulated Endothelial Cells.

In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA trans-Splicing In Vitro and in a Xenograft Model for Epidermolysis Bullosa-Associated Skin Cancer.

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

Light-Inducible Spatio-Temporal Control of TLR4 and NF-κB-Gluc Reporter in Human Pancreatic Cell Line.

Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options.

Concentration-Dependent Pro- and Antitumor Activities of Quercetin in Human Melanoma Spheroids: Comparative Analysis of 2D and 3D Cell Culture Models.

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

Cancer-type organic anion transporting polypeptide 1B3 is a target for cancer suicide gene therapy using RNA trans-splicing technology.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) has been identified as a cancer-specific transcript in various solid cancers, including colorectal cancer. Given its excellent cancer-specific expression profile, we hypothesized that Ct-OATP1B3 could represent a promising target for cancer-specific expression of the suicide gene, herpes simplex virus 1 thymidine kinase (HSV-tk), via a spliceosome-mediated RNA trans-splicing (SMaRT) approach. SMaRT technology is used to recombine two RNA molecules to generate a chimeric transcript. In this study, we engineered an RNA trans-splicing molecule carrying a translation-defective HSV-tk sequence (RTM44), which was capable of inducing its own trans-splicing to the desired Ct-OATP1B3 pre-mRNA target. RTM44 expression in LS180 cells resulted in generation of Ct-OATP1B3/HSV-tk fusion mRNA. A functional translation start site contributed by the target pre-mRNA restored HSV-tk protein expression, rendering LS180 cells sensitive to ganciclovir treatment in vitro and in xenografted mice. The observed effects are ascribed to accurate and efficient trans-splicing, as they were absent in cells carrying a splicing-deficient mutant of RTM44. Collectively, our data highlights Ct-OATP1B3 as an ideal target for the HSV-tk SMaRT suicide system, which opens up new translational avenues for Ct-OATP1B3-targeted cancer therapy.