The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

How Pyroptosis Contributes to Inflammation and Fibroblast-Macrophage Cross-Talk in Rheumatoid Arthritis.

About thirty years ago, a new form of pro-inflammatory lytic cell death was observed and termed pyroptosis. Only in 2015, gasdermins were defined as molecules that create pores at the plasma membrane and drive pyroptosis. Today, we know that gasdermin-mediated death is an important antimicrobial defence mechanism in bacteria, yeast and mammals as it destroys the intracellular niche for pathogen replication. However, excessive and uncontrolled cell death also contributes to immunopathology in several chronic inflammatory diseases, including arthritis. In this review, we discuss recent findings where pyroptosis contributes to tissue damage and inflammation with a main focus on injury-induced and autoimmune arthritis. We also review novel functions and regulatory mechanisms of the pyroptotic executors gasdermins. Finally, we discuss possible models of how pyroptosis may contribute to the cross-talk between fibroblast and macrophages, and also how this cross-talk may regulate inflammation by modulating inflammasome activation and pyroptosis induction.

Protein visualization and manipulation in Drosophila through the use of epitope tags recognized by nanobodies.

Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.

PHOrming the inflammasome: phosphorylation is a critical switch in inflammasome signalling.

Inflammasomes are protein complexes in the innate immune system that regulate the production of pro-inflammatory cytokines and inflammatory cell death. Inflammasome activation and subsequent cell death often occur within minutes to an hour, so the pathway must be dynamically controlled to prevent excessive inflammation and the development of inflammatory diseases. Phosphorylation is a fundamental post-translational modification that allows rapid control over protein function and the phosphorylation of inflammasome proteins has emerged as a key regulatory step in inflammasome activation. Phosphorylation of inflammasome sensor and adapter proteins regulates their inter- and intra-molecular interactions, subcellular localisation, and function. The control of inflammasome phosphorylation may thus provide a new strategy for the development of anti-inflammatory therapeutics. Herein we describe the current knowledge of how phosphorylation operates as a critical switch for inflammasome signalling.

TBK1 and IKKε act like an OFF switch to limit NLRP3 inflammasome pathway activation.

NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome activation is beneficial during infection and vaccination but, when uncontrolled, is detrimental and contributes to inflammation-driven pathologies. Hence, discovering endogenous mechanisms that regulate NLRP3 activation is important for disease interventions. Activation of NLRP3 is regulated at the transcriptional level and by posttranslational modifications. Here, we describe a posttranslational phospho-switch that licenses NLRP3 activation in macrophages. The ON switch is controlled by the protein phosphatase 2A (PP2A) downstream of a variety of NLRP3 activators in vitro and in lipopolysaccharide-induced peritonitis in vivo. The OFF switch is regulated by two closely related kinases, TANK-binding kinase 1 (TBK1) and I-kappa-B kinase epsilon (IKKε). Pharmacological inhibition of TBK1 and IKKε, as well as simultaneous deletion of TBK1 and IKKε, but not of either kinase alone, increases NLRP3 activation. In addition, TBK1/IKKε inhibitors counteract the effects of PP2A inhibition on inflammasome activity. We find that, mechanistically, TBK1 interacts with NLRP3 and controls the pathway activity at a site distinct from NLRP3-serine 3, previously reported to be under PP2A control. Mutagenesis of NLRP3 confirms serine 3 as an important phospho-switch site but, surprisingly, reveals that this is not the sole site regulated by either TBK1/IKKε or PP2A, because all retain the control over the NLRP3 pathway even when serine 3 is mutated. Altogether, a model emerges whereby TLR-activated TBK1 and IKKε act like a "parking brake" for NLRP3 activation at the time of priming, while PP2A helps remove this parking brake in the presence of NLRP3 activating signals, such as bacterial pore-forming toxins or endogenous danger signals.

Posttranslational and Therapeutic Control of Gasdermin-Mediated Pyroptosis and Inflammation.

Pyroptosis is a proinflammatory form of cell death, mediated by membrane pore-forming proteins called gasdermins. Gasdermin pores allow the release of the pro-inflammatory cytokines IL-1ß and IL-18 and cause cell swelling and cell lysis leading to release of other intracellular proteins that act as alarmins to perpetuate inflammation. The best characterized, gasdermin D, forms pores via its N-terminal domain, generated after the cleavage of full length gasdermin D by caspase-1 or -11 (caspase-4/5 in humans) typically upon sensing of intracellular pathogens. Thus, gasdermins were originally thought to largely contribute to pathogen-induced inflammation. We now know that gasdermin family members can also be cleaved by other proteases, such as caspase-3, caspase-8 and granzymes, and that they contribute to sterile inflammation as well as inflammation in autoinflammatory diseases or during cancer immunotherapy. Here we briefly review how and when gasdermin pores are formed, and then focus on emerging endogenous mechanisms and therapeutic approaches that could be used to control pore formation, pyroptosis and downstream inflammation.

Activation of a G protein-coupled receptor through indirect antibody-mediated tethering of ligands.

Antibodies raised against many cell surface proteins, including G protein-coupled receptors, remain important tools for their functional characterization. By linking antibodies to ligands for cell surface proteins, such adducts can be targeted to the surface of a cell type of choice. Site-specific functionalization of full-size antibodies with synthetic moieties remains challenging. Here we present new approaches in which single domain antibodies (known as VHHs or nanobodies) that target either cell surface proteins or conventional antibodies are used to indirectly deliver ligands for GPCRs to their sites of action. The combination of high yield production of nanobodies, facile site-specific functionalization, and compatibility with commercially available mouse and rabbit antibodies should enable wide application of this approach.

Improved GPCR ligands from nanobody tethering.

Antibodies conjugated to bioactive compounds allow targeted delivery of therapeutics to cell types of choice based on that antibody's specificity. Here we develop a new type of conjugate that consists of a nanobody and a peptidic ligand for a G protein-coupled receptor (GPCR), fused via their C-termini. We address activation of parathyroid hormone receptor-1 (PTHR1) and improve the signaling activity and specificity of otherwise poorly active N-terminal peptide fragments of PTH by conjugating them to nanobodies (VHHs) that recognize PTHR1. These C-to-C conjugates show biological activity superior to that of the parent fragment peptide in vitro. In an exploratory experiment in mice, a VHH-PTH peptide conjugate showed biological activity, whereas the corresponding free peptide did not. The lead conjugate also possesses selectivity for PTHR1 superior to that of PTH(1-34). This design approach, dubbed "conjugation of ligands and antibodies for membrane proteins" (CLAMP), can yield ligands with high potency and specificity.