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.

A role for the ITAM signaling module in specifying cytokine-receptor functions.

Diverse cellular responses to external cues are controlled by a small number of signal-transduction pathways, but how the specificity of functional outcomes is achieved remains unclear. Here we describe a mechanism for signal integration based on the functional coupling of two distinct signaling pathways widely used in leukocytes: the ITAM pathway and the Jak-STAT pathway. Through the use of the receptor for interferon-γ (IFN-γR) and the ITAM adaptor Fcγ as an example, we found that IFN-γ modified responses of the phagocytic antibody receptor FcγRI (CD64) to specify cell-autonomous antimicrobial functions. Unexpectedly, we also found that in peritoneal macrophages, IFN-γR itself required tonic signaling from Fcγ through the kinase PI(3)K for the induction of a subset of IFN-γ-specific antimicrobial functions. Our findings may be generalizable to other ITAM and Jak-STAT signaling pathways and may help explain signal integration by those pathways.

Inflammasomes as regulators of mechano-immunity.

Mechano-immunity, the intersection between cellular or tissue mechanics and immune cell function, is emerging as an important factor in many inflammatory diseases. Mechano-sensing defines how cells detect mechanical changes in their environment. Mechano-response defines how cells adapt to such changes, e.g. form synapses, signal or migrate. Inflammasomes are intracellular immune sensors that detect changes in tissue and cell homoeostasis during infection or injury. We and others recently found that mechano-sensing of tissue topology (swollen tissue), topography (presence and distribution of foreign solid implant) or biomechanics (stiffness), alters inflammasome activity. Once activated, inflammasomes induce the secretion of inflammatory cytokines, but also change cellular mechanical properties, which influence how cells move, change their shape, and interact with other cells. When overactive, inflammasomes lead to chronic inflammation. This clearly places inflammasomes as important players in mechano-immunity. Here, we discuss a model whereby inflammasomes integrate pathogen- and tissue-injury signals, with changes in tissue mechanics, to shape the downstream inflammatory responses and allow cell and tissue mechano-adaptation. We will review the emerging evidence that supports this model.

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.

Nur77 controls tolerance induction, terminal differentiation, and effector functions in semi-invariant natural killer T cells.

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77tg (Nur77tg;Vα14tg) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4-producing NKT2 cell subset but not IFN-γ-producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions.

Ciliary proteins specify the cell inflammatory response by tuning NFκB signalling, independently of primary cilia.

Complex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting the potential for these pathways to be targeted therapeutically. Primary cilia are subcellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1ß, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviours, changing cytosolic NFκB translocation dynamics but leaving MAPK signalling unaffected. RNA-seq analysis indicates that IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf Through microscopy, we find altered NFκB dynamics are independent of assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits inflammatory responses in the non-ciliated macrophage. We propose that ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme to tune cytoplasmic NFκB signalling and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation.This article has an associated First Person interview with the first author of the paper.

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.

Integration of cytokine and heterologous receptor signaling pathways.

Cytokines are soluble mediators of cell communication that are critical in immune regulation. They induce specific gene-expression programs in responsive cells. Recent findings, however, indicate that cytokine receptors can regulate immune cell functions by transcription-independent mechanisms. Here we review the current understanding of how cytokine signaling regulates the functions of other signaling pathways by first discussing the 'traditional' transcription-mediated consequences of cytokine signaling and then providing a detailed description of transcription-independent lateral communications between cytokine receptors and other cellular receptors.

Cutting Edge: Blockade of Inhibitor of Apoptosis Proteins Sensitizes Neutrophils to TNF- but Not Lipopolysaccharide-Mediated Cell Death and IL-1ß Secretion.

The mammalian inhibitor of apoptosis proteins (IAPs) are key regulators of cell death and inflammation. A major function of IAPs is to block the formation of a cell death-inducing complex, termed the ripoptosome, which can trigger caspase-8-dependent apoptosis or caspase-independent necroptosis. Recent studies report that upon TLR4 or TNF receptor 1 (TNFR1) signaling in macrophages, the ripoptosome can also induce NLRP3 inflammasome formation and IL-1ß maturation. Whether neutrophils have the capacity to assemble a ripoptosome to induce cell death and inflammasome activation during TLR4 and TNFR1 signaling is unclear. In this study, we demonstrate that murine neutrophils can signal via TNFR1-driven ripoptosome assembly to induce both cell death and IL-1ß maturation. However, unlike macrophages, neutrophils suppress TLR4-dependent cell death and NLRP3 inflammasome activation during IAP inhibition via deficiencies in the CD14/TRIF arm of TLR4 signaling.

NKG2D signaling is coupled to the interleukin 15 receptor signaling pathway.

The effector functions of natural killer cells are regulated by activating receptors, which recognize stress-inducible ligands expressed on target cells and signal through association with signaling adaptors. Here we developed a mouse model in which a fusion of the signaling adaptor DAP10 and ubiquitin efficiently downregulated expression of the activating receptor NKG2D on the surfaces of natural killer cells. With this system, we identified coupling of the signaling pathways triggered by NKG2D and DAP10 to those initiated by the interleukin 15 receptor. We suggest that this coupling of activating receptors to other receptor systems could function more generally to regulate cell type-specific signaling events in distinct physiological contexts.

The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists.

Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL-1ß production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL-1ß production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum-induced IL-1ß production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response.

Signaling pathways activated by a protease allergen in basophils.

Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain's immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils.

Role for lysosomal phospholipase A2 in iNKT cell-mediated CD1d recognition.

Invariant natural killer T (iNKT) cells recognize self lipid antigens presented by CD1d molecules. The nature of the self-antigens involved in the development and maturation of iNKT cells is poorly defined. Lysophospholipids are self-antigens presented by CD1d that are generated through the action of phospholipases A1 and A2. Lysosomal phospholipase A2 (LPLA2, group XV phospholipase A2) resides in the endocytic system, the main site where CD1d antigen acquisition occurs, suggesting that it could be particularly important in CD1d function. We find that Lpla2(-/-) mice show a decrease in iNKT cell numbers that is neither the result of a general effect on the development of lymphocyte populations nor of effects on CD1d expression. However, endogenous lipid antigen presentation by CD1d is reduced in the absence of LPLA2. Our data suggest that LPLA2 plays a role in the generation of CD1d complexes with thymic lipids required for the normal selection and maturation of iNKT cells.

IL-15 regulates homeostasis and terminal maturation of NKT cells.

Semi-invariant NKT cells are thymus-derived innate-like lymphocytes that modulate microbial and tumor immunity as well as autoimmune diseases. These immunoregulatory properties of NKT cells are acquired during their development. Much has been learned regarding the molecular and cellular cues that promote NKT cell development, yet how these cells are maintained in the thymus and the periphery and how they acquire functional competence are incompletely understood. We found that IL-15 induced several Bcl-2 family survival factors in thymic and splenic NKT cells in vitro. Yet, IL-15-mediated thymic and peripheral NKT cell survival critically depended on Bcl-x(L) expression. Additionally, IL-15 regulated thymic developmental stage 2 to stage 3 lineage progression and terminal NKT cell differentiation. Global gene expression analyses and validation revealed that IL-15 regulated Tbx21 (T-bet) expression in thymic NKT cells. The loss of IL-15 also resulted in poor expression of key effector molecules such as IFN-γ, granzyme A and C, as well as several NK cell receptors, which are also regulated by T-bet in NKT cells. Taken together, our findings reveal a critical role for IL-15 in NKT cell survival, which is mediated by Bcl-x(L), and effector differentiation, which is consistent with a role of T-bet in regulating terminal maturation.

Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms.

Adjuvanted protein vaccines offer high efficacy, yet most potent adjuvants remain proprietary. Several adjuvant compounds are being developed by the Vaccine Formulation Institute in Switzerland for global open access clinical use. In the context of the R21 malaria vaccine, in a mouse challenge model, we characterize the efficacy and mechanism of action of four Vaccine Formulation Institute adjuvants: two liposomal (LQ and LMQ) and two squalene emulsion-based adjuvants (SQ and SMQ), containing QS-21 saponin (Q) and optionally a synthetic TLR4 agonist (M). Two R21 vaccine formulations, R21/LMQ and R21/SQ, offer the highest protection (81%-100%), yet they trigger different innate sensing mechanisms in macrophages with LMQ, but not SQ, activating the NLRP3 inflammasome. The resulting in vivo adaptive responses have a different TH1/TH2 balance and engage divergent innate pathways while retaining high protective efficacy. We describe how modular changes in vaccine formulation allow for the dissection of the underlying immune pathways, enabling future mechanistically informed vaccine design.

Activation of the Non-canonical Inflammasome in Mouse and Human Cells.

The non-canonical inflammasome is a signaling platform that allows for the detection of cytoplasmic lipopolysaccharides (LPS) in immune and non-immune cells. Upon detection of LPS, this inflammasome activates the signaling proteases caspase-4 and -5 (in humans) and caspase-11 (in mice). Inflammatory caspases activation leads to caspase self-processing and the cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD N-terminal fragments oligomerize and form pores at the plasma membranes, leading to an inflammatory form of cell death called pyroptosis. Here, we describe a simple method to activate the non-canonical inflammasome in myeloid and epithelial cells and to measure its activity using cell death assay and immunoblotting.

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.

B cell-intrinsic TBK1 is essential for germinal center formation during infection and vaccination in mice.

The germinal center (GC) is a site where somatic hypermutation and clonal selection are coupled for antibody affinity maturation against infections. However, how GCs are formed and regulated is incompletely understood. Here, we identified an unexpected role of Tank-binding kinase-1 (TBK1) as a crucial B cell-intrinsic factor for GC formation. Using immunization and malaria infection models, we show that TBK1-deficient B cells failed to form GC despite normal Tfh cell differentiation, although some malaria-infected B cell-specific TBK1-deficient mice could survive by GC-independent mechanisms. Mechanistically, TBK1 phosphorylation elevates in B cells during GC differentiation and regulates the balance of IRF4/BCL6 expression by limiting CD40 and BCR activation through noncanonical NF-κB and AKTT308 signaling. In the absence of TBK1, CD40 and BCR signaling synergistically enhanced IRF4 expression in Pre-GC, leading to BCL6 suppression, and therefore failed to form GCs. As a result, memory B cells generated from TBK1-deficient B cells fail to confer sterile immunity upon reinfection, suggesting that TBK1 determines B cell fate to promote long-lasting humoral immunity.

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.

NKT Cells Join the Two Step for Inflammasome-Independent IL-1ß Release.

The cytokine interleukin-1ß (IL-1ß) is critical for antimicrobial defenses; the inflammasome pathway typically controls IL-1ß release, but pathogens often evade this pathway. In this issue, Donado et al. (2020) describe an alternative, two-cell model, to instruct inflammasome-independent IL-1ß release.