Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1? production in response to ?-glucans and the fungal pathogen, Candida albicans.
ABSTRACT: Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1? maturation and resistance to fungal dissemination in Candida albicans infection. ?-Glucans are major components of fungal cell walls that trigger IL-1? secretion in both murine and human immune cells. In this study, we sought to determine the contribution of ?-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1? production in response to ?-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating ?-glucan-induced IL-1? processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting ?-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1? maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1? production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating ?-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between ?-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.
Project description:Candida albicans is an opportunistic fungal pathogen causing life-threatening mucosal and systemic infections in immunocompromised humans. Using a murine model of mucosal Candida infection, we investigated the role of the proinflammatory cytokine IL-1beta in host defense to Candida albicans. We find that the synthesis, processing, and release of IL-1beta in response to Candida are tightly controlled and first require transcriptional induction, followed by a second signal leading to caspase-1-mediated cleavage of the pro-IL-1beta cytokine. The known fungal pattern recognition receptors TLR2 and Dectin-1 regulate IL-1beta gene transcription, whereas the NLRP3-containing proinflammatory multiprotein complex, the NLRP3 inflammasome, controls caspase-1-mediated cleavage of pro-IL-1beta. Furthermore, we show that TLR2, Dectin-1, and NLRP3 are essential for defense against dissemination of mucosal infection and mortality in vivo. Therefore, in addition to sensing bacterial and viral pathogens, the NLRP3 inflammasome senses fungal pathogens and is critical in host defense against Candida.
Project description:The NLRP3 inflammasome is activated in response to different bacterial, viral, and fungal pathogens and serves as modulator of different pattern recognition receptors signaling pathways. One of the main functions of NLRP3 is to participate in IL-1? maturation which is important in the host defense against Pneumocystis and other fungal infections. However, dysregulation of NLRP3 and IL-1? secretion are also implicated in the pathophysiology of many auto-inflammatory disorders. Often time's inflammatory flares are preceded by infectious illnesses questioning the role of infection in autoimmune exacerbations. However, we still do not fully understand the exact role that infection or even colonization plays as a trigger of inflammation. Herein, we investigated the role of NLRP3 in circulating B-lymphocytes following activation with two major microbial antigens (?-glucan and CpG). NLRP3 was determined essential in two independent B-lymphocytes processes: pro-inflammatory cytokine secretion and antibody regulation. Our results show that the ?-glucan fungal cell wall carbohydrate stimulated B-lymphocytes to secrete IL-1? in a process partially mediated by Dectin-1 activation via SYK and the transcription factors NF-?B and AP-1. This IL-1? secretion was regulated by the NLRP3 inflammasome and was dependent on potassium efflux and Caspase-1. Interestingly, B-lymphocytes activated by unmethylated CpG motifs, found in bacterial and fungal DNA, failed to induce IL-1?. However, B-lymphocyte stimulation by CpG resulted in NLRP3 and Caspase-1 activation and the production and secretion of IgM antibodies. Furthermore, CpG-stimulated IgM secretion, unlike ?-glucan-mediated IL-1? production, was mediated by the mammalian target of rapamycin (mTOR). Inhibition of NLRP3 and the mTOR pathway in CpG activated B-lymphocytes resulted in impaired IgM secretion suggesting their participation in antibody regulation. In conclusion, this study describes a differential response of NLRP3 to ?-glucan and CpG antigens and identifies the NLRP3 inflammasome of human circulating B-lymphocytes as a modulator of the innate and adaptive immune systems.
Project description:Invasive aspergillosis (IA) is a life-threatening disease that occurs in immunodepressed patients when infected with Aspergillus fumigatus. This fungus is the second most-common causative agent of fungal disease after Candida albicans. Nevertheless, much remains to be learned about the mechanisms by which A. fulmigatus activates the innate immune system. We investigated the inflammatory response to conidia and hyphae of A. fumigatus and specifically, their capacity to trigger activation of an inflammasome. Our results show that in contrast to conidia, hyphal fragments induce NLRP3 inflammasome assembly, caspase-1 activation and IL-1beta release from a human monocyte cell line. The ability of Aspergillus hyphae to activate the NLRP3 inflammasome in the monocytes requires K(+) efflux and ROS production. In addition, our data show that NLRP3 inflammasome activation as well as pro-IL-1beta expression relies on the Syk tyrosine kinase, which is downstream from the pathogen recognition receptor Dectin-1, reinforcing the importance of Dectin-1 in the innate immune response against fungal infection. Furthermore, we show that treatment of monocytes with corticosteroids inhibits transcription of the gene encoding IL-1beta. Thus, our data demonstrate that the innate immune response against A. fumigatus infection involves a two step activation process, with a first signal promoting expression and synthesis of pro-IL-1beta; and a second signal, involving Syk-induced activation of the NLRP3 inflammasome and caspase-1, allowing processing and secretion of the mature cytokine.
Project description:Recognition of the fungal cell wall carbohydrate ?-glucan by the host receptor Dectin-1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal ?-glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat-killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin-dependent phagocytosis of particulate (1-3,1-6)-?-glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal ?-glucan particles upon actin cytoskeleton disruption, relies on Dectin-1 and results in the classical caspase-1 inflammasome formation through NLRP3, generation of an oxidative burst, NF-?B activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections.
Project description:Inflammasome is an intracellular protein complex that serves as cytosolic pattern recognition receptor (PRR) to engage with pathogens and to process cytokines of the interleukin-1 (IL-1) family into bioactive molecules. It has been established that interleukin-1? (IL-1?) is important to host defense against Histoplasma capsulatum infection. However, the detailed mechanism of how H. capsulatum induces inflammasome activation leading to IL-1? production has not been studied. Here, we showed in dendritic cells (DCs) that H. capsulatum triggers caspase-1 activation and IL-1? production through NLRP3 inflammasome. By reciprocal blocking of Dectin-1 or Dectin-2 in single receptor-deficient DCs and cells from Clec4n-/-, Clec7a-/-, and Clec7a-/-Clec4n-/- mice, we discovered that while Dectin-2 operates as a primary receptor, Dectin-1 serves as a secondary one for NLRP3 inflammasome. In addition, both receptors trigger Syk-JNK signal pathway to activate signal 1 (pro-IL-1? synthesis) and signal 2 (activation of caspase-1). Results of pulmonary infection with H. capsulatum showed that CD103+ DCs are one of the major producers of IL-1? and Dectin-2 and Dectin-1 double deficiency abolishes their IL-1? response to the fungus. While K+ efflux and cathepsin B (but not ROS) function as signal 2, viable but not heat-killed H. capsulatum triggers profound lysosomal rupture leading to cathepsin B release. Interestingly, cathepsin B release is regulated by ERK/JNK downstream of Dectin-2 and Dectin-1. Our study demonstrates for the first time the unique roles of Dectin-2 and Dectin-1 in triggering Syk-JNK to activate signal 1 and 2 for H. capsulatum-induced NLRP3 inflammasome activation.
Project description:Numerous atypical mycobacteria, including Mycobacterium abscessus (Mabc), cause nontuberculous mycobacterial infections, which present a serious public health threat. Inflammasome activation is involved in host defense and the pathogenesis of autoimmune diseases. However, inflammasome activation has not been widely characterized in human macrophages infected with atypical mycobacteria. Here, we demonstrate that Mabc robustly activates the nucleotide binding and oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome via dectin-1/Syk-dependent signaling and the cytoplasmic scaffold protein p62/SQSTM1 (p62) in human macrophages. Both dectin-1 and Toll-like receptor 2 (TLR2) were required for Mabc-induced mRNA expression of pro-interleukin (IL)-1?, cathelicidin human cationic antimicrobial protein-18/LL-37 and ?-defensin 4 (DEFB4). Dectin-1-dependent Syk signaling, but not that of MyD88, led to the activation of caspase-1 and secretion of IL-1? through the activation of an NLRP3/apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) inflammasome. Additionally, potassium efflux was required for Mabc-induced NLRP3/ASC inflammasome activation. Furthermore, Mabc-induced p62 expression was critically involved in NLRP3 inflammasome activation in human macrophages. Finally, NLRP3/ASC was critical for the inflammasome in antimicrobial responses to Mabc infection. Taken together, these data demonstrate the induction mechanism of the NLRP3/ASC inflammasome and its role in innate immunity to Mabc infection.
Project description:Extrapolating from lessons learnt with previous low-molecular-weight ?-(1?3)-glucan mimetics, we designed a series of minimal 2,4-dideoxy-thioether-linked carbacyclic ?-(1?3)-glucan mimetics and synthesized di-, tri-, and tetramers in an enantiomerically pure form by an iterative sequence based on a simple building block readily available from commercial ( S)-(-)-3-cyclohexenecarboxylic acid. These substances were screened for their ability to inhibit anti-CR3-fluorescein isothiocyanate (FITC) staining of human neutrophils and anti-Dectin-1-FITC staining of mouse macrophages as well as for their ability to stimulate phagocytosis and pinocytosis. In each assay, the synthetic compounds displayed comparable activity to the corresponding native ?-(1?3)-glucans, laminaritriose, and tetraose, suggesting that the exploitation of hydrophobic patches in the lectin-binding domains of CR3 and Dectin-1 is a promising strategy for the development of small-molecule analogues of ?-(1?3)-glucans.
Project description:The immunomodulatory properties of yeast ?-1,3/1,6 glucans are mediated through their ability to be recognized by human innate immune cells. While several studies have investigated binding of opsonized and unopsonized particulate ?-glucans to human immune cells mainly via complement receptor 3 (CR3) or Dectin-1, few have focused on understanding the binding characteristics of soluble ?-glucans. Using a well-characterized, pharmaceutical-grade, soluble yeast ?-glucan, this study evaluated and characterized the binding of soluble ?-glucan to human neutrophils and monocytes. The results demonstrated that soluble ?-glucan bound to both human neutrophils and monocytes in a concentration-dependent and receptor-specific manner. Antibodies blocking the CD11b and CD18 chains of CR3 significantly inhibited binding to both cell types, establishing CR3 as the key receptor recognizing the soluble ?-glucan in these cells. Binding of soluble ?-glucan to human neutrophils and monocytes required serum and was also dependent on incubation time and temperature, strongly suggesting that binding was complement-mediated. Indeed, binding was reduced in heat-inactivated serum, or in serum treated with methylamine or in serum reacted with the C3-specific inhibitor compstatin. Opsonization of soluble ?-glucan was demonstrated by detection of iC3b, the complement opsonin on ?-glucan-bound cells, as well as by the direct binding of iC3b to ?-glucan in the absence of cells. Binding of ?-glucan to cells was partially inhibited by blockade of the alternative pathway of complement, suggesting that the C3 activation amplification step mediated by this pathway also contributed to binding.
Project description:The pathogenic yeast Candida albicans escapes macrophages by triggering NLRP3 inflammasome-dependent host cell death (pyroptosis). Pyroptosis is inflammatory and must be tightly regulated by host and microbe, but the mechanism is incompletely defined. We characterized the C. albicans endoplasmic reticulum (ER)-mitochondrion tether ERMES and show that the ERMES mmm1 mutant is severely crippled in killing macrophages despite hyphal formation and normal phagocytosis and survival. To understand dynamic inflammasome responses to Candida with high spatiotemporal resolution, we established live-cell imaging for parallel detection of inflammasome activation and pyroptosis at the single-cell level. This showed that the inflammasome response to mmm1 mutant hyphae is delayed by 10 h, after which an exacerbated activation occurs. The NLRP3 inhibitor MCC950 inhibited inflammasome activation and pyroptosis by C. albicans, including exacerbated inflammasome activation by the mmm1 mutant. At the cell biology level, inactivation of ERMES led to a rapid collapse of mitochondrial tubular morphology, slow growth and hyphal elongation at host temperature, and reduced exposed 1,3-β-glucan in hyphal populations. Our data suggest that inflammasome activation by C. albicans requires a signal threshold dependent on hyphal elongation and cell wall remodeling, which could fine-tune the response relative to the level of danger posed by C. albicans. The phenotypes of the ERMES mutant and the lack of conservation in animals suggest that ERMES is a promising antifungal drug target. Our data further indicate that NLRP3 inhibition by MCC950 could modulate C. albicans-induced inflammation. IMPORTANCE The yeast Candida albicans causes human infections that have mortality rates approaching 50%. The key to developing improved therapeutics is to understand the host-pathogen interface. A critical interaction is that with macrophages: intracellular Candida triggers the NLRP3/caspase-1 inflammasome for escape through lytic host cell death, but this also activates antifungal responses. To better understand how the inflammasome response to Candida is fine-tuned, we established live-cell imaging of inflammasome activation at single-cell resolution, coupled with analysis of the fungal ERMES complex, a mitochondrial regulator that lacks human homologs. We show that ERMES mediates Candida escape via inflammasome-dependent processes, and our data suggest that inflammasome activation is controlled by the level of hyphal growth and exposure of cell wall components as a proxy for severity of danger. Our study provides the most detailed dynamic analysis of inflammasome responses to a fungal pathogen so far and establishes promising pathogen- and host-derived therapeutic strategies.
Project description:The murine molecule dectin-1 (known as the beta-glucan receptor in humans) is an immune cell surface receptor implicated in the immunological defense against fungal pathogens. Sequence analysis has indicated that the dectin-1 extracellular domain is a C-type lectin-like domain, and functional studies have established that it binds fungal beta-glucans. We report several dectin-1 crystal structures, including a high-resolution structure and a 2.8 angstroms resolution structure in which a short soaked natural beta-glucan is trapped in the crystal lattice. In vitro characterization of dectin-1 in the presence of its natural ligand indicates higher-order complex formation between dectin-1 and beta-glucans. These combined structural and biophysical data considerably extend the current knowledge of dectin-1 structure and function, and suggest potential mechanisms of defense against fungal pathogens.