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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.
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.
Role of ITAM signaling module in signal integration.
Diverse cell types use a small number of evolutionarily conserved signaling modules to integrate external cues and elicit distinct functions. A question thus arises as to how does a receptor, which contains a single signaling module, produce distinct outcomes to diverse signals, particularly if such module is shared amongst a family of receptors? Emerging data suggest that many immunoreceptors, all of which use a conserved ITAM-module for their signaling, can couple with members of additional classes of membrane receptors to deliver unique signal(s) to the cell. We discuss the possible biological purposes and mechanisms behind these interactions at the plasma membrane. We offer a conceptual framework to understand information processing within the immune system and discuss the new biology of old receptors involving their structural and functional collaborations that evolved to deliver unique signal(s) to the cell using a limited set of conserved signaling modules.
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.
Sterile signals generate weaker and delayed macrophage NLRP3 inflammasome responses relative to microbial signals.
Inflammation is the host response to microbial infection or sterile injury that aims to eliminate the insult, repair the tissue and restore homeostasis. Macrophages and the NLRP3 inflammasome are key sentinels for both types of insult. Although it is well established that the NLRP3 inflammasome is activated by microbial products and molecules released during sterile injury, it is unclear whether the responses elicited by these different types of signals are distinct. In this study, we used lipopolysaccharide and tumor necrosis factor as prototypical microbial and sterile signal 1 stimuli, respectively, to prime the NLRP3 inflammasome. We then used the bacterial toxin nigericin and a common product released from necrotic cells, ATP, as prototypical microbial and sterile signal 2 stimuli, respectively, to trigger the assembly of the NLRP3 inflammasome complex in mouse and human macrophages. We found that NLRP3 inflammasome responses were weakest when both signal 1 and signal 2 were sterile, but responses were faster and stronger when at least one of the two signals was microbial. Ultimately, the most rapid and potent responses were elicited when both signals were microbial. Together, these data suggest that microbial versus sterile signals are distinct, both kinetically and in magnitude, in their ability to generate inflammasome-dependent responses. This hierarchy of NLRP3 responses to sterile versus microbial stimuli likely reflects the urgent need for the immune system to respond rapidly to the presence of infection to halt pathogen dissemination.
A Staphylococcus aureus regulatory system that responds to host heme and modulates virulence.
Staphylococcus aureus, a bacterium responsible for tremendous morbidity and mortality, exists as a harmless commensal in approximately 25% of humans. Identifying the molecular machinery activated upon infection is central to understanding staphylococcal pathogenesis. We describe the heme sensor system (HssRS) that responds to heme exposure and activates expression of the heme-regulated transporter (HrtAB). Inactivation of the Hss or Hrt systems leads to increased virulence in a vertebrate infection model, a phenotype that is associated with an inhibited innate immune response. We suggest that the coordinated activity of Hss and Hrt allows S. aureus to sense internal host tissues, resulting in tempered virulence to avoid excessive host tissue damage. Further, genomic analyses have identified orthologous Hss and Hrt systems in Bacillus anthracis, Listeria monocytogenes, and Enterococcus faecalis, suggesting a conserved regulatory system by which Gram-positive pathogens sense heme as a molecular marker of internal host tissue and modulate virulence.
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.
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.
Quantitative and qualitative differences in the in vivo response of NKT cells to distinct alpha- and beta-anomeric glycolipids.
NKT cells represent a unique subset of immunoregulatory T cells that recognize glycolipid Ags presented by the MHC class I-like molecule CD1d. Because of their immunoregulatory properties, NKT cells are attractive targets for the development of immunotherapies. The prototypical NKT cell ligand alpha-galactosylceramide (alpha-GalCer), originally isolated from a marine sponge, has potent immunomodulatory activities in mice, demonstrating therapeutic efficacy against metastatic tumors, infections, and autoimmune diseases, but also has a number of adverse side effects. In vivo administration of alpha-GalCer to mice results in the rapid activation of NKT cells, which is characterized by cytokine secretion, surface receptor down-regulation, expansion, and secondary activation of a variety of innate and adaptive immune system cells. In this study, we have evaluated the in vivo immune response of mice to a set of structural analogues of alpha-GalCer. Our results show that, contrary to current thinking, beta-anomeric GalCer can induce CD1d-dependent biological activities in mice, albeit at lower potency than alpha-anomeric GalCer. In addition, we show that the response of NKT cells to distinct GalCer differs not only quantitatively, but also qualitatively. These findings indicate that NKT cells can fine-tune their immune responses to distinct glycolipid Ags in vivo, a property that may be exploited for the development of effective and safe NKT cell-based immunotherapies.
Distinct roles of dendritic cells and B cells in Va14Ja18 natural T cell activation in vivo.
Va14Ja18 natural T (iNKT) cells are innate, immunoregulatory lymphocytes that recognize CD1d-restricted lipid Ags such as alpha-galactosylceramide (alpha GalCer). The immunoregulatory functions of iNKT cells are dependent upon either IFN-gamma or IL-4 production by these cells. We hypothesized that alpha GalCer presentation by different CD1d-positive cell types elicits distinct iNKT cell functions. In this study we report that dendritic cells (DC) play a critical role in alpha GalCer-mediated activation of iNKT cells and subsequent transactivation of NK cells. Remarkably, B lymphocytes suppress DC-mediated iNKT and NK cell activation. Nevertheless, alpha GalCer presentation by B cells elicits low IL-4 responses from iNKT cells. This finding is particularly interesting because we demonstrate that NOD DC are defective in eliciting iNKT cell function, but their B cells preferentially activate this T cell subset to secrete low levels of IL-4. Thus, the differential immune outcome based on the type of APC that displays glycolipid Ags in vivo has implications for the design of therapies that harness the immunoregulatory functions of iNKT cells.
Cutting edge: the ontogeny and function of Va14Ja18 natural T lymphocytes require signal processing by protein kinase C theta and NF-kappa B.
The rapid and robust immunoregulatory cytokine response of Va14Ja18 natural T (iNKT) cells to glycolipid Ags determines their diverse functions. Unlike conventional T cells, iNKT lymphocyte ontogeny absolutely requires NF-kappa B signaling. However, the precise role of NF-kappa B in iNKT cell function and the identity of upstream signals that activate NF-kappa B in this T cell subset remain unknown. Using mice in which iNKT cell ontogeny has been rescued despite inhibition of NF-kappa B signaling, we demonstrate that iNKT cell function requires NF-kappa B in a lymphocyte-intrinsic manner. Furthermore, the ontogeny of functional iNKT cells requires signaling through protein kinase C theta, which is dispensable for conventional T lymphocyte development. The unique requirement of protein kinase C theta implies that signals emanating from the TCR activate NF-kappa B during iNKT cell development and function. Thus, we conclude that NF-kappa B signaling plays a crucial role at distinct levels of iNKT cell biology.
NF-kappa B controls cell fate specification, survival, and molecular differentiation of immunoregulatory natural T lymphocytes.
Ontogenetic, homeostatic, and functional deficiencies within immunoregulatory natural T (iNKT) lymphocytes underlie various inflammatory immune disorders including autoimmunity. Signaling events that control cell fate specification and molecular differentiation of iNKT cells are only partly understood. Here we demonstrate that these processes within iNKT cells require classical NF-kappaB signaling. Inhibition of NF-kappaB signaling blocks iNKT cell ontogeny at an immature stage and reveals an apparent, novel precursor in which negative selection occurs. Most importantly, this block occurs due to a lack of survival signals, as Bcl-x(L) overexpression rescues iNKT cell ontogeny. Maturation of immature iNKT cell precursors induces Bcl-2 expression, which is defective in the absence of NF-kappaB signaling. Bcl-x(L) overexpression also rescues this maturation-induced Bcl-2 expression. Thus, antiapoptotic signals relayed by NF-kappaB critically control cell fate specification and molecular differentiation of iNKT cells and, hence, reveal a novel role for such signals within the immune system.
Commitment toward the natural T (iNKT) cell lineage occurs at the CD4+8+ stage of thymic ontogeny.
T lineage commitment occurs in a discrete, stage-specific manner during thymic ontogeny. Intrathymic precursor transfer experiments and the identification of CD4(+)8+ double-positive (DP), V alpha 14J alpha 18 natural T (iNKT) cells suggest that commitment to this lineage might occur at the DP stage. Nevertheless, this matter remains contentious because others failed to detect V alpha 14J alpha 18-positive iNKT cells that are CD4(+)8+. In resolution to this issue, we demonstrate that retinoic acid receptor-related orphan receptor gamma (ROR gamma)0/0 thymi, which accumulate immature single-positive (ISP) thymocytes that precede the DP stage, do not rearrange V alpha 14-to-J alpha 18 gene segments, suggesting that this event occurs at a post-ISP stage. Mixed radiation bone marrow chimeras revealed that RORgamma functions in an iNKT cell lineage-specific manner. Further, introgression of a Bcl-x(L) transgene into ROR gamma(0/0) mice, which promotes survival and permits secondary rearrangements of distal V alpha and J alpha gene segments at the DP stage, rescues V alpha 14-to-J alpha 18 recombination. Similarly, introgression of a rearranged V alpha 14J alpha 18 transgene into ROR gamma(0/0) mice results in functional iNKT cells. Thus, our data support the "T cell receptor-instructive (mainstream precursor) model" of iNKT cell lineage specification where V alpha 14-to-J alpha 18 rearrangement, positive selection, and iNKT cell lineage commitment occur at or after the DP stage of ontogeny.
Another view of T cell antigen recognition: cooperative engagement of glycolipid antigens by Va14Ja18 natural T(iNKT) cell receptor [corrected].
Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell function are partly defined. However, the impact of iNKT cell receptor beta-chain repertoire and how alpha-galactosylceramide (alpha-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-alphaGalCer- and alphaGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional alphabeta TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.
Natural killer T cells accelerate atherogenesis in mice.
We have investigated the potential role of CD1d-restricted natural killer T (NKT) cells in the development of atherosclerosis in mice. When fed an atherogenic diet (AD), NKT cell-deficient CD1d(-/-) mice had significantly smaller atherosclerotic lesions than AD-fed C57BL/6 (wild-type [WT]) mice. A significant reduction in atherosclerotic lesions was also demonstrated in AD-fed, low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice reconstituted with CD1d(-/-) bone marrow cells compared with the lesions observed in Ldlr(-/-)mice reconstituted with WT marrow cells. In addition, repeated injections of alpha-GalCer or the related glycolipid OCH to apolipoprotein E knockout (apoE(-/-)) mice during the early phase of atherosclerosis significantly enlarged the lesion areas compared with mice injected with vehicle control. However, administering alpha-GalCer to apoE(-/-) mice with established lesions did not significantly increase the lesion area but considerably decreased the collagen content. Atherosclerosis development in either AD-fed WT or apoE(-/-) mice was associated with the presence of Valpha14Jalpha18 transcripts in the atherosclerotic arterial walls, indicating that NKT cells were recruited to these lesions. Thioglycolate-elicited macrophages pulsed with oxidized low-density lipoproteins expressed enhanced CD1d levels and induced NKT cells to produce interferon-gamma, a potentially proatherogenic T-helper 1 (TH1) cytokine. Collectively, we conclude that NKT cells are proatherogenic in mice.
Increase in hepatic NKT cells in leukocyte cell-derived chemotaxin 2-deficient mice contributes to severe concanavalin A-induced hepatitis.
Leukocyte cell-derived chemotaxin 2 (LECT2) was originally identified for its possible chemotactic activity against human neutrophils in vitro. It is a 16-kDa protein that is preferentially expressed in the liver. Its homologues have been widely identified in many vertebrates. Current evidence suggests that LECT2 may be a multifunctional protein like cytokines. However, the function of LECT2 in vivo remains unclear. To elucidate the role of this protein in vivo, we have generated LECT2-deficient (LECT2(-/-)) mice. We found that the proportion of NKT cells in the liver increased significantly in LECT2(-/-) mice, although those of conventional T cells, NK cells, and other cell types were comparable with those in wild-type mice. Consistent with increased hepatic NKT cell number, the production of IL-4 and IFN-gamma was augmented in LECT2(-/-) mice upon stimulation with alpha-galactosylceramide, which specifically activates Valpha14 NKT cells. In addition, NKT cell-mediated cytotoxic activity against syngeneic thymocytes increased in hepatic mononuclear cells obtained from LECT2(-/-) mice in vitro. Interestingly, the hepatic injury was exacerbated in LECT2(-/-) mice upon treatment with Con A, possibly because of the significantly higher expression of IL-4 and Fas ligand. These results suggest that LECT2 might regulate the homeostasis of NKT cells in the liver and might be involved in the pathogenesis of hepatitis.
Characterization and Functional Analysis of Mouse Semi-invariant Natural T Cells.
Semi-invariant natural killer T (iNKT) cells are CD1d-restricted innate-like lymphocytes that recognize lipid agonists. Activated iNKT cells have immunoregulatory properties. Human and mouse iNKT cell functions elicited by different glycolipid agonists are highly conserved, making the mouse an excellent animal model for understanding iNKT cell biology in vivo. This unit describes basic methods for the characterization and quantification (see Basic Protocol 1) and functional analysis of mouse iNKT cells in vivo or in vitro. This unit also contains protocols that describe enrichment and purification of iNKT cells, generation of CD1d tetramer, and lipid antigen loading onto cell-bound and soluble CD1d for activation of NKT cell hybridomas. © 2017 by John Wiley & Sons, Inc.
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.
Ciliary proteins specify the cell inflammatory response by tuning NFκB signaling, independently of primary cilia.
Complex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting these pathways as therapeutic targets. Primary cilia are sub-cellular 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-1β, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviors, changing cytosolic NFκB translocation dynamics, but leaving MAPK unaffected. RNAseq analysis indicates IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf. By microscopy, we find altered NFκB dynamics are independent to assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits the inflammatory responses in the non-ciliated macrophage. We propose 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.
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.