Methods to Investigate the Role of Toll-Like Receptors in Allergic Contact Dermatitis
Allergic contact disease is a common inflammatory skin disease resulting from hyperresponsiveness to harmless nonprotein substances such as metals, fragrances, or rubber. Recent research has highlighted a prominent role of Toll-like receptors, particularly TLR4 in contact allergen-induced innate immune activation that crucially contributes to the pathogenesis of this disease. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Understanding the Role of Cellular Molecular Clocks in Controlling the Innate Immune Response
The importance of the 24-h daily cycle, termed circadian, on immune function has been highlighted by a number of recent studies. Immune parameters such as the response to bacterial challenge or immune cell trafficking change with time of day and disruption of circadian rhythms has been linked to inflammatory pathologies. We are beginning to uncover that the key proteins that comprise the molecular clock, most notably BMAL1, CLOCK, and REV-ERBα, also control fundamental aspects of the immune response. Given the ubiquitous nature of the molecular clock in controlling many other types of physiologies such as metabolism ...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Toll-Like Receptor Signalling and the Control of Intestinal Barrier Function
Epithelial barrier function and innate immunity are fundamental to the pathogenesis of inflammatory and infectious disease. Along with plasma membranes, epithelial cells are the primary cellular determinant of epithelial barrier function. The mechanism by which polarized epithelia form a permeability barrier is of fundamental importance to the prevention of many infectious and inflammatory diseases. Moreover, epithelial cells express Toll-like receptors (TLRs) which upon recognition of conserved microbial factors such as lipopolysaccharide (LPS) induce epithelial responses including epithelial cell proliferation, secretion...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Analysis of TLR-Induced Metabolic Changes in Dendritic Cells Using the Seahorse XFe96 Extracellular Flux Analyzer
Engagement of Toll-like receptors (TLRs) on dendritic cells (DCs) triggers the expression of a large set of genes involved in DC activation and maturation, which allow them to act efficiently as antigen-presenting cells. Recently, it has become clear that TLR signalling in DCs also results in dramatic metabolic changes that are integral to their changed biology. Here, we describe a detailed protocol on how DC metabolism can be studied after TLR stimulation using the 96-well format Extracellular Flux (XFe96) Analyzer from Seahorse Bioscience, a machine that allows one to simultaneously assess rates of oxidative phosphorylat...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Toll-Like Receptor-Dependent Immune Complex Activation of B Cells and Dendritic Cells
High titers of autoantibodies reactive with DNA/RNA molecular complexes are characteristic of autoimmune disorders such as systemic lupus erythematosus (SLE). In vitro and in vivo studies have implicated the endosomal Toll-like receptor 9 (TLR9) and Toll-like receptor 7 (TLR7) in the activation of the corresponding autoantibody producing B cells. Importantly, TLR9/TLR7-deficiency results in the inability of autoreactive B cells to proliferate in response to DNA/RNA-associated autoantigens in vitro, and in marked changes in the autoantibody repertoire of autoimmune-prone mice. Uptake of DNA/RNA-associated autoantigen immune...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Analysis by Flow Cytometry of B-Cell Activation and Antibody Responses Induced by Toll-Like Receptors
Toll-like receptors (TLRs) are expressed in B lymphocytes and contribute to B-cell activation, antibody responses, and their maturation. TLR stimulation of mouse B cells induces class switch DNA recombination (CSR) to isotypes specified by cytokines, and also induces formation of IgM+ as well as class-switched plasma cells. B-cell receptor (BCR) signaling, while on its own inducing limited B-cell proliferation and no CSR, can enhance CSR driven by TLRs. Particular synergistic or antagonistic interactions among TLR pathways, BCR, and cytokine signaling can have important consequences for B-cell activation, CSR, and plasma c...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

TLR Function in Murine CD4+ T Lymphocytes and Their Role in Inflammation
Toll-like receptor (TLR) signaling represents an evolutionary-conserved mechanism allowing for the rapid detection of broad molecular patterns that are common to different groups of pathogens. TLRs are traditionally associated with cells of the innate immune response where ligation of a TLR alone can lead to cellular activation and the initialization of an immune response. Cells of adaptive immunity, namely different classes of T and B lymphocytes, are also known to express a variety of TLRs. Conversely, the functional and signaling outcomes of TLRs are decidedly different in cells of the adaptive immune response. T lympho...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Analysis of Post-transcriptional Gene Regulation of Nod-Like Receptors via the 3′UTR
Innate immune signaling is the front line of defense against pathogens, leading to an appropriate response of immune cells upon activation of their pattern recognition receptors (PRRs) by microbial products, such as Toll-like receptors (TLRs). Apart from transcriptional control, gene expression in the innate immune system is also highly regulated at the post-transcriptional level. miRNA or RNA-binding protein can bind to the 3′ untranslated region (UTR) of target mRNAs and affect their mRNA stability and translation efficiency, which ultimately affects the amount of protein that is produced. In recent years, a new gr...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Determining the Function of Long Noncoding RNA in Innate Immunity
The advent of deep sequencing technologies has provided us with an unprecedented view of the human genome. Over 85 % of the genome is actively transcribed, yet we do not know the function of the vast majority of these RNA transcripts. Long noncoding RNAs (lncRNA) represent the largest group of RNA genes transcribed in the cell and currently there is limited experimental data supporting the functions of a very small proportion of these transcripts. lncRNA are expressed in a highly cell type specific manner and our interests involve understanding the role they play in innate immune signaling networks. In this chapter I will ...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Simple Methods to Investigate MicroRNA Induction in Response to Toll-Like Receptors
In this chapter, we describe simple methods to investigate microRNA (miRNA) induction in response to lipopolysaccharide, the ligand for Toll-Like Receptor-4 activation. In brief, we demonstrate how to investigate global miRNA induction and/or repression in bone marrow-derived macrophages using TaqMan MicroRNA Arrays, followed by methods to measure individual miRNAs and target mRNA expression. Moreover, we explain step-by-step instructions on how to modulate endogenous miRNA expression through the use of miRNA inhibitors and mimics as well as highlight how miRNA modulation can be used to confirm mRNA targeting via Luciferas...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Co-transcriptomic Analysis by RNA Sequencing to Simultaneously Measure Regulated Gene Expression in Host and Bacterial Pathogen
Intramacrophage pathogens subvert antimicrobial defence pathways using various mechanisms, including the targeting of host TLR-mediated transcriptional responses. Conversely, TLR-inducible host defence mechanisms subject intramacrophage pathogens to stress, thus altering pathogen gene expression programs. Important biological insights can thus be gained through the analysis of gene expression changes in both the host and the pathogen during an infection. Traditionally, research methods have involved the use of qPCR, microarrays and/or RNA sequencing to identify transcriptional changes in either the host or the pathogen. He...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Measuring Monomer-to-Filament Transition of MAVS as an In Vitro Activity Assay for RIG-I-Like Receptors
During viral infection, the innate immune RIG-I like receptors (RLRs) recognize viral double stranded RNA (dsRNA) and trigger filament assembly of the adaptor protein Mitochondrial Anti-viral Signaling protein (MAVS). The MAVS filament then activates anti-viral signaling events including the up-regulation of type I interferon expression. In recent years, much insight has been gained into how RLRs recognize dsRNA, but the precise mechanism of how activated RLRs stimulate MAVS filament formation remains less understood. In this chapter, we describe an in vitro reconstitution assay that we have previously developed to study t...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity
In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads and cells can be measured by flow cytometry, which results in an easily quantifiable readout. Furthermore, it allows measuring of binding by specific cell subsets within a mixed cell population. Overall, this method is a convenient and easily standardized assay for measuring binding. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Detection of Interaction Between Toll-Like Receptors and Other Transmembrane Proteins by Co-immunoprecipitation Assay
Toll-like receptors are type I membrane proteins and bind other membrane proteins often via a specific interaction between transmembrane domains. The co-immunoprecipitation assay is a widely used biochemical technique for assessing interactions among proteins in cell lysates or tissue extracts. By isolating a native protein complex with a specific antibody against a protein of interest, followed by western blotting with an antibody for a binding partner, the co-immunoprecipitation assay can be used to confirm a putative interaction between two proteins. The co-immunoprecipitation assay can also be combined with a proteomic...
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news

Methods for Delivering DNA to Intracellular Receptors
We describe here optimized procedures for both electroporation and chemical transfection, including the centrifugation of chemical transfection reagent onto cells, which greatly increases the speed and strength of responses. Appropriate choice of DNA and use of these methods allow study of either the cytosolic DNA responses in isolation or the simultaneous stimulation of cytosolic receptors and the CpG DNA receptor toll-like receptor 9 (TLR9) in the endosomes. (Source: Springer protocols feed by Immunology)
Source: Springer protocols feed by Immunology - January 7, 2016 Category: Allergy & Immunology Source Type: news