Interaction of TLR4 and TLR8 in the Innate Immune Response against Mycobacterium Tuberculosis.

The interaction and crosstalk of Toll-like receptors (TLRs) is an established pathway in which the innate immune system recognises and fights pathogens. In a single nucleotide polymorphisms (SNP) analysis of an Indian cohort, we found evidence for both TLR4-399T and TRL8-1A conveying increased susceptibility towards tuberculosis (TB) in an interdependent manner, even though there is no established TLR4 ligand present in Mycobacterium tuberculosis (Mtb), which is the causative pathogen of TB. Docking studies revealed that TLR4 and TLR8 can build a heterodimer, allowing interaction with TLR8 ligands. The conformational change of TLR4-399T might impair this interaction. With immunoprecipitation and mass spectrometry, we precipitated TLR4 with TLR8-targeted antibodies, indicating heterodimerisation. Confocal microscopy confirmed a high co-localisation frequency of TLR4 and TLR8 that further increased upon TLR8 stimulation. The heterodimerisation of TLR4 and TLR8 led to an induction of IL12p40, NF-κB, and IRF3. TLR4-399T in interaction with TLR8 induced an increased NF-κB response as compared to TLR4-399C, which was potentially caused by an alteration of subsequent immunological pathways involving type I IFNs. In summary, we present evidence that the heterodimerisation of TLR4 and TLR8 at the endosome is involved in Mtb recognition via TLR8 ligands, such as microbial RNA, which induces a Th1 response. These findings may lead to novel targets for therapeutic interventions and vaccine development regarding TB.

Macrophage frequency in the bone marrow correlates with morphologic subtype of myeloproliferative neoplasm.

Bone marrow (BM) fibrosis in myeloproliferative neoplasms (MPNs) is associated with a poor prognosis. The development of myelofibrosis and differentiation of mesenchymal stromal cells to profibrotic myofibroblasts depends on macrophages. Here, we compared macrophage frequencies in BM biopsies of MPN patients and controls (patients with non-neoplastic processes), including primary myelofibrosis (PMF, n = 18), essential thrombocythemia (ET, n = 14), polycythemia vera (PV, n = 12), and Philadelphia chromosome-positive chronic myeloid leukemia (CML, n = 9). In PMF, CD68-positive macrophages were greatly increased compared to CML (p = 0.017) and control BM (p < 0.001). Similar findings were observed by CD163 staining (PMF vs. CML: p = 0.017; PMF vs. control: p < 0.001). Moreover, CD68-positive macrophages were increased in PV compared with ET (p = 0.009) and reactive cases (p < 0.001). PMF had higher frequencies of macrophages than PV (CD68: p < 0.001; CD163: p < 0.001) and ET (CD68: p < 0.001; CD163: p < 0.001). CD163 and CD68 were often co-expressed in macrophages with stellate morphology in Philadelphia chromosome-negative MPN, resulting in a sponge-like reticular network that may be a key regulator of unbalanced hematopoiesis in the BM space and may explain differences in cellularity and clinical course.

NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain.

NLRP3 is a cytosolic pattern recognition receptor that senses microbes and endogenous danger signals. Upon activation, NLRP3 forms an inflammasome with the adapter ASC, resulting in caspase-1 activation, release of proinflammatory cytokines and cell death. How NLRP3 activation is regulated by transcriptional and posttranslational mechanisms to prevent aberrant activation remains incompletely understood. Here, we identify three conserved phosphorylation sites in NLRP3 and demonstrate that NLRP3 activation is controlled by phosphorylation of its pyrin domain (PYD). Phosphomimetic residues in NLRP3 PYD abrogate inflammasome activation and structural modeling indicates that phosphorylation of the PYD regulates charge-charge interaction between two PYDs that are essential for NLRP3 activation. Phosphatase 2A (PP2A) inhibition or knock-down drastically reduces NLRP3 activation, showing that PP2A can license inflammasome assembly via dephosphorylating NLRP3 PYD. These results propose that the balance between kinases and phosphatases acting on the NLRP3 PYD is critical for NLRP3 activation.

RAGE Enhances TLR Responses through Binding and Internalization of RNA.

Nucleic acid recognition is an important mechanism that enables the innate immune system to detect microbial infection and tissue damage. To minimize the recognition of self-derived nucleic acids, all nucleic acid-sensing signaling receptors are sequestered away from the cell surface and are activated in the cytoplasm or in endosomes. Nucleic acid sensing in endosomes relies on members of the TLR family. The receptor for advanced glycation end-products (RAGE) was recently shown to bind DNA at the cell surface, facilitating DNA internalization and subsequent recognition by TLR9. In this article, we show that RAGE binds RNA molecules in a sequence-independent manner and enhances cellular RNA uptake into endosomes. Gain- and loss-of-function studies demonstrate that RAGE increases the sensitivity of all ssRNA-sensing TLRs (TLR7, TLR8, TLR13), suggesting that RAGE is an integral part of the endosomal nucleic acid-sensing system.

Automated nanoscale flow cytometry for assessing protein-protein interactions.

Despite their importance for signalling events, protein-protein interactions cannot easily be analyzed on a single cell level. We developed a robust automated FRET measurement system implemented on a commercial flow cytometer allowing for rapid profiling of molecular associations in living cells. We used this method to measure the most proximal signaling events on human T lymphocyte activation, which preceded calcium influx, and could automatically detect T cell receptor/CD3 complex clustering defects in immunocompromised patients. © 2016 International Society for Advancement of Cytometry.

Toll-Like Receptor Interactions Measured by Microscopic and Flow Cytometric FRET.

Protein-protein interactions regulate biological networks. The most proximal events that initiate signal transduction frequently are receptor dimerization or conformational changes in receptor complexes. Toll-like receptors (TLRs) are transmembrane receptors that are activated by a number of exogenous and endogenous ligands. Most TLRs can respond to multiple ligands and the different TLRs recognize structurally diverse molecules ranging from proteins, sugars, lipids, and nucleic acids. TLRs can be expressed on the plasma membrane or in endosomal compartments and ligand recognition thus proceeds in different microenvironments. Not surprisingly, distinctive mechanisms of TLR receptor activation have evolved. A detailed understanding of the mechanisms of TLR activation is important for the development of novel synthetic TLR activators or pharmacological inhibitors of TLRs. Confocal laser scanning microscopy combined with GFP technology allows the direct visualization of TLR expression in living cells. Fluorescence resonance energy transfer (FRET) measurements between two differentially tagged proteins permit the study of TLR interaction, and distances between receptors in the range of molecular interactions can be measured and visualized. Additionally, FRET measurements combined with confocal microscopy provide detailed information about molecular interactions in different subcellular localizations. These techniques permit the dynamic visualization of early signaling events in living cells and can be utilized in pharmacological or genetic screens.

[Tl(III)(dota)](-): An Extraordinarily Robust Macrocyclic Complex.

The X-ray structure of {C(NH2)3}[Tl(dota)]·H2O shows that the Tl(3+) ion is deeply buried in the macrocyclic cavity of the dota(4-) ligand (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) with average Tl-N and Tl-O distances of 2.464 and 2.365 Å, respectively. The metal ion is directly coordinated to the eight donor atoms of the ligand, which results in a twisted square antiprismatic (TSAP') coordination around Tl(3+). A multinuclear (1)H, (13)C, and (205)Tl NMR study combined with DFT calculations confirmed the TSAP' structure of the complex in aqueous solution, which exists as the Λ(λλλλ)/Δ(δδδδ) enantiomeric pair. (205)Tl NMR spectroscopy allowed the protonation constant associated with the protonation of the complex according to [Tl(dota)](-) + H(+) ⇆ [Tl(Hdota)] to be determined, which turned out to be pK(H)Tl(dota) = 1.4 ± 0.1. [Tl(dota)](-) does not react with Br(-), even when using an excess of the anion, but it forms a weak mixed complex with cyanide, [Tl(dota)](-) + CN(-) ⇆ [Tl(dota)(CN)](2-), with an equilibrium constant of Kmix = 6.0 ± 0.8. The dissociation of the [Tl(dota)](-) complex was determined by UV-vis spectrophotometry under acidic conditions using a large excess of Br(-), and it was found to follow proton-assisted kinetics and to take place very slowly (∼10 days), even in 1 M HClO4, with the estimated half-life of the process being in the 10(9) h range at neutral pH. The solution dynamics of [Tl(dota)](-) were investigated using (13)C NMR spectroscopy and DFT calculations. The (13)C NMR spectra recorded at low temperature (272 K) point to C4 symmetry of the complex in solution, which averages to C4v as the temperature increases. This dynamic behavior was attributed to the Λ(λλλλ) ↔ Δ(δδδδ) enantiomerization process, which involves both the inversion of the macrocyclic unit and the rotation of the pendant arms. According to our calculations, the arm-rotation process limits the Λ(λλλλ) ↔ Δ(δδδδ) interconversion.

Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP.

The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2'-5'). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2'-5') is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2'-5') transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.

RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA.

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

ASC speck formation as a readout for inflammasome activation.

All inflammasomes require the adapter protein apoptosis associated speck-like protein containing a CARD (ASC) for the activation of caspase-1. After inflammasome activation, ASC assembles into a large protein complex, which is termed "speck". ASC specks can be observed as they reach a size of around 1 µm and in most cells only one speck forms upon inflammasome activation. Hence, ASC speck formation can be used as a simple upstream readout for inflammasome activation. Here, we describe a method for analyzing inflammasome activation by ASC speck visualization. First, we describe the generation of a clonal inflammasome reporter macrophage cell line overexpressing fluorescently tagged ASC. We then discuss stimulation conditions and the microscopic evaluation of ASC speck formation.

Intracellular sensing of microbes and danger signals by the inflammasomes.

The cells of the innate immune system mobilize a coordinated immune response towards invading microbes and after disturbances in tissue homeostasis. These immune responses typically lead to infection control and tissue repair. Exaggerated or uncontrolled immune responses, however, can also induce acute of chronic inflammatory pathologies that are characteristic for many common diseases such as sepsis, arthritis, atherosclerosis, or Alzheimer's disease. In recent years, the concerted efforts of many scientists have uncovered numerous mechanisms by which immune cells detect foreign or changed self-substances that appear in infections or during tissue damage. These substances stimulate signaling receptors, which leads to cellular activation and the induction of effector mechanisms. Here, we review the role of inflammasomes, a family of signaling molecules that form multi-molecular signaling platforms and activate inflammatory caspases and interleukin-1ß cytokines.