Allergen recognition by innate immune cells: critical role of dendritic and epithelial cells

Allergy is an exacerbated response of the immune system against non-self-proteins called allergens and is typically characterized by biased type-2 T helper cell and deleterious IgE mediated immune responses. The allergic cascade starts with the recognition of allergens by antigen presenting cells, mainly dendritic cells (DCs), leading to Th2 polarization, switching to IgE production by B cells, culminating in mast cell sensitization and triggering. DCs have been demonstrated to play a crucial role in orchestrating allergic diseases. Using different C-type lectin receptors DCs are able to recognize and internalize a number of allergens from diverse sources leading to sensitization. Furthermore, there is increasing evidence highlighting the role of epithelial cells in triggering and modulating immune responses to allergens. As well as providing a physical barrier, epithelial cells can interact with allergens and influence DCs behavior through the release of a number of Th2 promoting cytokines. In this review we will summarize current understanding of how allergens are recognized by DCs and epithelial cells and what are the consequences of such interaction in the context of allergic sensitization and downstream events leading to allergic inflammation. Better understanding of the molecular mechanisms of allergen recognition and associated signaling pathways could enable developing more effective therapeutic strategies that target the initial steps of allergic sensitization hence hindering development or progression of allergic diseases

Holographic optical trapping Raman micro-spectroscopy for non-invasive measurement and manipulation of live cells

We present a new approach for combining holographic optical tweezers with confocal Raman spectroscopy. Multiple laser foci, generated using a liquid-crystal spatial light modulator, are individually used for both optical trapping and excitation of spontaneous Raman spectroscopy from trapped objects. Raman scattering from each laser focus is spatially filtered using reflective apertures on a digital micro-mirror device, which can be reconfigured with flexible patterns at video rate. We discuss operation of the instrument, and performance and viability considerations for biological measurements. We then demonstrate the capability of the instrument for fast, flexible, and interactive manipulation with molecular measurement of interacting live cell systems

Tissue transglutaminase treatment leads to concentration-dependent changes in dendritic cell phenotype--implications for the role of transglutaminase in coeliac disease.

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Dendritic cells (DCs) are part of the innate immune system with a key role in initiating and modulating T cell mediated immune responses. Coeliac disease is caused by inappropriate activation of such a response leading to small intestinal inflammation when gluten is ingested. Tissue transglutaminase, an extracellular matrix (ECM) protein, has an established role in coeliac disease; however, little work to date has examined its impact on DCs. The aim of this study was to investigate the effect of small intestinal ECM proteins, fibronectin (FN) and tissue transglutaminase 2 (TG-2), on human DCs by including these proteins in DC cultures.The study used flow cytometry and scanning electron microscopy to determine the effect of FN and TG-2 on phenotype, endocytic ability and and morphology of DCs. Furthermore, DCs treated with FN and TG-2 were cultured with T cells and subsequent T cell proliferation and cytokine profile was determined.The data indicate that transglutaminase affected DCs in a concentration-dependent manner. High concentrations were associated with a more mature phenotype and increased ability to stimulate T cells, while lower concentrations led to maintenance of an immature phenotype.These data provide support for an additional role for transglutaminase in coeliac disease and demonstrate the potential of in vitro modelling of coeliac disease pathogenesis.Published versio

Metabolic characterisation of THP-1 macrophage polarisation using LC–MS-based metabolite profiling

INTRODUCTION: Macrophages constitute a heterogeneous population of functionally distinct cells involved in several physiological and pathological processes. They display remarkable plasticity by changing their phenotype and function in response to environmental cues representing a spectrum of different functional phenotypes. The so-called M1 and M2 macrophages are often considered as representative of pro- and anti-inflammatory ends of such spectrum. Metabolomics approach is a powerful tool providing important chemical information about the cellular phenotype of living systems, and the changes in their metabolic pathways in response to various perturbations. OBJECTIVES: This study aimed to characterise M1 and M2 phenotypes in THP-1 macrophagesin order to identify characteristic metabolites of each polarisation state. METHODS: Herein, untargeted liquid chromatography (LC)-mass spectrometry (MS)-based metabolite profiling was applied to characterise the metabolic profile of M1-like and M2-like THP-1 macrophages. RESULTS: The results showed that M1 and M2 macrophages have distinct metabolic profiles. Sphingolipid and pyrimidine metabolism was significantly changed in M1 macrophages whereas arginine, proline, alanine, aspartate and glutamate metabolism was significantly altered in M2 macrophages. CONCLUSION: This study represents successful application of LC-MS metabolomics approach to characterise M1 and M2 macrophages providing functional readouts that show unique metabolic signature for each phenotype. These data could contribute to a better understanding of M1 and M2 functional properties and could pave the way for developing new therapeutics targeting different immune diseases

The role of indoleamine 2,3-dioxygenase-aryl hydrocarbon receptor pathway in the TLR4-induced tolerogenic phenotype in human DCs

A controlled inflammatory response is required for protection against infection, but persistent inflammation causes tissue damage. Dendritic cells (DCs) have a unique capacity to promote both inflammatory and anti-inflammatory processes. One key mechanism involved in DC-mediated immunosuppression is the expression of tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO has been implicated in diverse processes in health and disease but its role in endotoxin tolerance in human DCs is still controversial. Here we investigated the role of IDO in shaping DCs phenotype and function under endotoxin tolerance conditions. Our data show that TLR4 ligation in LPS-primed DCs, induced higher levels of both IDO isoforms together with the transcription factor aryl-hydrocarbon receptor (AhR), compared to unprimed controls. Additionally, LPS conditioning induced an anti-inflammatory phenotype in DCs - with an increase in IL-10 and higher expression of programmed death ligand (PD-L)1 and PD-L2 - which were partially dependent on IDO. Furthermore, we demonstrated that the AhR-IDO pathway was responsible for the preferential activation of noncanonical NF-κB pathway in LPS-conditioned DCs. These data provide new insight into the mechanisms of the TLR4-induced tolerogenic phenotype in human DCs, which can help the better understanding of processes involved in induction and resolution of chronic inflammation and tolerance

Editorial: Adverse reactions to biomaterials: State of the art in biomaterial risk assessment, immunomodulation and in vitro models for biomaterial testing

Editorial on the Research Topi

Analysis of proteomic profiles and functional properties of human peripheral blood myeloid dendritic cells, monocyte-derived dendritic cells and the dendritic cell-like KG-1 cells reveals distinct characteristics

Important proteomic and functional differences between peripheral blood myeloid dendritic cells, monocyte-derived dendritic cells (moDC) and KG-1 cells have been identified

Role of ROCK isoforms in regulation of stiffness induced myofibroblast differentiation in lung fibrosis

Fibrosis is a major cause of progressive organ dysfunction in several chronic pulmonary diseases. Rho associated coiled-coil forming kinase (ROCK) has shown to be involved in myofibroblast differentiation driven by altered matrix stiffness in fibrotic state. There are two known ROCK isoforms in human, ROCK1 (ROKβ) and ROCK2 (ROKα), but specific role of each isoform in myofibroblast differentiation in lung fibrosis remains unknown. To study this, we developed a Gelatin methacryloyl (GelMA) hydrogel based culture system with different stiffness levels relevant to healthy and fibrotic lungs. We have shown that stiff matrix and not soft matrix, can induce myofibroblast differentiation with high αSMA expression. Furthermore, our data confirm that the inhibition of ROCK signalling by a pharmacological inhibitor (i.e. Y27632) attenuates stiffness induced αSMA expression and fibre assembly in myofibroblasts. To assess the role of ROCK isoforms in this process we used siRNA to knock down the expression of each isoform. Our data showed that knocking down either ROCK1 or ROCK2 did not result in a reduction in αSMA expression in myofibroblasts on stiff matrix as opposed to soft matrix where αSMA expression was reduced significantly. Paradoxically, on stiff matrix, the absence of one isoform (particularly ROCK2) exaggerated αSMA expression and led to thick fibre assembly. Moreover complete loss of αSMA fibre assembly was seen only in the absence of both ROCK isoforms suggesting that both isoforms are implicated in this process. Overall our results indicate the differential role of ROCK isoforms in myofibroblast differentiation on soft and stiff matrices

Developing immune-regulatory materials using immobilized monosaccharides with immune-instructive properties

New strategies for immune modulation have shown real promise in regenerative medicine as well as the fight against autoimmune diseases, allergies and cancer. Dendritic cells (DCs) are gatekeepers of the immune system and their ability in shaping the adaptive immune responses makes DCs ideal targets for immune modulation. Carbohydrates are abundant in different biological systems and are known to modulate DC phenotype and function. However, how simple monosaccharides instruct DC function is less well understood. In this study we used a combinatorial array of immobilized monosaccharides to investigate how they modulate DC phenotype and function, and crucially the impact of such changes on downstream adaptive immune responses. Our data show a selection of monosaccharides significantly suppress lipopolysaccharide (LPS) induced DC activation as evidenced by reduction in CD40 expression, IL-12 production and 2,3 indoleamine dioxygenase activity, while inducing a significant increase in IL-10 production. These changes are indicative of induction of an anti-inflammatory or regulatory phenotype in DCs which was further confirmed in DC-T cell co-cultures where DCs cultured on the ‘regulatory’ monosaccharaide coated surfaces were shown to induce naïve T cell polarization towards regulatory phenotype. Our data also highlighted a selection of monosaccharides that are able to promote mixed Treg and Th17 cell differentiation, a T cell phenotype expected to be highly immune suppressive. These data show the potential immunomodulatory effects of immobilized monosaccharides in priming DCs and skewing T cell differentiation towards an immune-regulatory phenotype. The ability to fine tune immune responses using these simple carbohydrate combinations (e.g. as coatings for existing materials) can be utilized as novel tools for immune modulation with potential applications in regenerative medicine, implantable medical devices and wound healing where reduction of inflammatory responses and maintaining immune homeostasis are desirable

New generation of bioreactors that advance extracellular matrix modelling and tissue engineering

Bioreactors hold a lot of promise for tissue engineering and regenerative medicine applications. They have multiple uses including cell cultivation for therapeutic production and for in vitro organ modelling to provide a more physiologically relevant environment for cultures compared to conventional static conditions. Bioreactors are often used in combination with scaffolds as the nutrient flow can enhance oxygen and diffusion throughout the 3D constructs to prevent the formation of necrotic cores. A variety of scaffolds have been fabricated to achieve a structural architecture that mimic native extracellular matrix. Future developments of in vitro models will incorporate the ability to non-invasively monitor the cellular microenvironment to enhance the understanding of in vitro conditions. This review details current advancements in bioreactor and scaffold systems and provides insight on how in vitro models can be augmented for future biomedical applications