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Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence
Abstract
Fungal infections represent a serious threat, particularly in immunocompromised patients. Interleukin-1beta (IL-1beta) is a key pro-inflammatory factor in innate antifungal immunity. The mechanism by which the mammalian immune system regulates IL-1beta production after fungal recognition is unclear. Two signals are generally required for IL-1beta production: an NF-kappaB-dependent signal that induces the synthesis of pro-IL-1beta (p35), and a second signal that triggers proteolytic pro-IL-1beta processing to produce bioactive IL-1beta (p17) via Caspase-1-containing multiprotein complexes called inflammasomes. Here we demonstrate that the tyrosine kinase Syk, operating downstream of several immunoreceptor tyrosine-based activation motif (ITAM)-coupled fungal pattern recognition receptors, controls both pro-IL-1beta synthesis and inflammasome activation after cell stimulation with Candida albicans. Whereas Syk signalling for pro-IL-1beta synthesis selectively uses the Card9 pathway, inflammasome activation by the fungus involves reactive oxygen species production and potassium efflux. Genetic deletion or pharmalogical inhibition of Syk selectively abrogated inflammasome activation by C. albicans but not by inflammasome activators such as Salmonella typhimurium or the bacterial toxin nigericin. Nlrp3 (also known as NALP3) was identified as the critical NOD-like receptor family member that transduces the fungal recognition signal to the inflammasome adaptor Asc (Pycard) for Caspase-1 (Casp1) activation and pro-IL-1beta processing. Consistent with an essential role for Nlrp3 inflammasomes in antifungal immunity, we show that Nlrp3-deficient mice are hypersusceptible to Candida albicans infection. Thus, our results demonstrate the molecular basis for IL-1beta production after fungal infection and identify a crucial function for the Nlrp3 inflammasome in mammalian host defence in vivo.
... Inflammasomes are intracellular multiprotein complexes that sense danger signals from damaged cells and pathogens. Mice lacking the NLRP3 inflammasome components, NLRP3, ASC, and caspase-1, are highly susceptible to systemic candidiasis [21][22][23] . The caspase-1dependent cytokines, IL-1β and IL-18, are required for the initiation of the protective Th1 and Th17 cellular responses 22,24 . ...
... β-glucans, the most abundant cell wall component of fungi, are recognized by cell surface receptors, such as Dectin-1 and CR3, and cytoplasmic inflammasome components, resulting in proinflammatory gene transcription and NLRP3 inflammasome activation [26][27][28] . C. albicans hyphae activate the NLRP3 inflammasome significantly more than yeasts, leading to increased production of IL-1β 21,23,29,30 . Candidalysin and Saps also trigger NLRP3 inflammasome activation 31,32 . ...
... Syk kinase signaling is critical for the host defense against fungal pathogens 23 . Immunoblotting of WT M1 BMDMs after C. albicans infection showed that both the control strain and the als1 SY als3 SY als5 Δ/Δ mutant induced robust Syk phosphorylation that persisted for 1 hour (Fig. 4d). ...
Patients with decreased levels of CD18 (β2 integrins) suffer from life-threatening bacterial and fungal infections. CD11b, the α subunit of integrin CR3 (CD11b/CD18, αMβ2), is essential for mice to fight against systemic Candida albicans infections. Live elongating C. albicans activates CR3 in immune cells. However, the hyphal ligands that activate CR3 are not well defined. Here, we discovered that the C. albicans Als family proteins are recognized by the I domain of CD11b in macrophages. This recognition synergizes with the β-glucan-bound lectin-like domain to activate CR3, thereby promoting Syk signaling and inflammasome activation. Dectin-2 activation serves as the “outside-in signaling” for CR3 activation at the entry site of incompletely sealed phagosomes, where a thick cuff of F-actin forms to strengthen the local interaction. In vitro, CD18 partially contributes to IL-1β release from dendritic cells induced by purified hyphal Als3. In vivo, Als3 is vital for C. albicans clearance in mouse kidneys. These findings uncover a novel family of ligands for the CR3 I domain that promotes fungal clearance.
... In a last step, Ninj1 mediates plasma membrane rupture causing a lytic form of cell death [33] called pyroptosis. In experimental settings, purified substances only trigger priming/ licensing or NLRP3 activation, but typically not both, while live pathogens can provide both signals simultaneously [34]. An exception is certain imidazoquinolines such as imiquimod (R837) that can induce priming through TLR7/8 as well as NLRP3 activation involving mitochondrial electron transport chain (ETC) complex I inhibition [35]. ...
... 14. Alternative priming signals that are occasionally used in the literature are R848 (80 μM), heat-killed Saccharomyces cerevisiae or Candida albicans [34], or the purified yeast cell wall components zymosan and mannan [57] as well as the algaederived β-glucan curdlan. ...
... MSU crystals can also be self-prepared as previously described [58]. 34. For aluminum hydroxide, it has to be mentioned that the term "Alum" that is commonly used as a synonym for crystals of non-soluble aluminum salts that activate the NLRP3 inflammasome is actually imprecise. ...
The inflammasome-nucleating cytoplasmic sensor protein NLRP3 (NACHT-, LRR, and PYD domains-containing protein 3, also known as NOD-like receptor pyrin domain-containing 3, NALP3, or cryopyrin) is triggered by a broad spectrum of sterile endogenous danger signals and environmental irritants. Upon activation, NLRP3 engages the adapter protein ASC that in turn recruits the third inflammasome component, the protease caspase-1. Subsequent caspase-1 activation leads to its auto-processing and maturation of the leaderless IL-1 family cytokines IL-1β and IL-18 as well as cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD plasma membrane pores, formed by its N-terminus, facilitate IL-1 release and, typically, subsequent cell lysis (pyroptosis). This protocol explains standard methods, which are routinely used in our laboratory to study NLRP3 inflammasome biology in vitro. It includes experimental approaches using primary murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs), human peripheral blood mononuclear cells (PBMCs), as well as inflammasome-competent cell lines (HoxB8 and THP-1 cells). The protocol covers the use of a broad spectrum of established NLRP3 activators and outlines the use of common inhibitors blocking NLRP3 itself or its upstream triggering events. We also provide guidelines for experimental set-up and crucial experimental controls to investigate NLRP3 inflammasome signaling or study new activators and inhibitors.
... CLRs activate Syk and CARD9 mediated signaling pathways to induce the production of various inflammatory cytokines to clear C. albicans [9]. Nlrp3, a member of NLRs, recognizes C. albicans and activates inflammasomes to mediate IL-1β production, which activates monocytes, macrophages, and neutrophils and induces Th1 and Th17 responses to fight against fungal infection [10,11]. In addition, Nlrc4 and Nlrp3 synergistically recruit more neutrophils to the infection sites by activating inflammasomes, which promotes Th17-mediated immunity and the production of antibacterial peptides to eliminate pathogens during oral C. albicans infection [12]. ...
... BMDMs were cocultured with C. albicans (MOI: 10) or treated with SMT (400 μ M, Beyotime). One hour later, amphotericin B was added to kill the extracellular fungus for 1 h before washing. ...
Candida albicans is a common opportunistic pathogenic fungus. The innate immune system provides the first-line host defense against fungal infection. Innate immune receptors and downstream molecules have been shown to play various roles during fungal infection. The innate immune receptor MDA5, encoded by the gene Ifih1, enhances host resistance against viral and Aspergillus fumigatus infection by inducing the production of interferons (IFNs). However, the role of MDA5 in C. albicans infection is still unclear. Here, we found that the gene expression levels of IFIH1 were significantly increased in innate immune cells after C. albicans stimulation through human bioinformatics analysis or mouse experiments. Through in vivo study, MDA5 was shown to enhance host susceptibility to C. albicans infection independent of IFN production. Instead, MDA5 exerted its influence on macrophages and kidneys by modulating the expression of Noxa, Bcl2, and Bax, thereby promoting apoptosis. Additionally, MDA5 compromised killing capabilities of macrophage by inhibition iNOS expression. The introduction of the apoptosis inducer PAC1 further impaired macrophage functions, mimicking the enhancing effect of MDA5 on C. albicans infection. Furthermore, the administration of macrophage scavengers increased the susceptibility of Ifih1−/− mice to C. albicans. The founding suggests that MDA5 promote host susceptibility to invasive C. albicans by enhancing cell apoptosis and compromising macrophage functions, making MDA5 a target to treat candidiasis.
... NLRP3 inflammasome can be activated not only by a variety of endogenous dangerous Signaling molecule and environmental stimuli (DAMP), but also by a variety of pathogen components or products (PAMP), such as Nigericin, Candida albicans, Staphylococcus aureus, etc. [99]. Nigericin is a polyether antibiotic derived from a metabolite of the grampositive bacterium S.hygroscopicus [100]. It is often used as a K + efflux carrier to promote K + /H + exchange across biofilms, as an effective research tool, it can activate ATPase activity and destroy mitochondrial Membrane potential [101]. ...
... K + efflux is considered to be the basic mechanism of ATPinduced NLRP3 inflammasome activation, and K efflux has become a common mechanism of NLRP3 inflammasome activation [111]. Most or all NLRP3 inducers (including ATP, nigricin, and silica) induce K + efflux and promote the activation of the NLRP3 inflammasome when K + effluxes in the cytoplasm are reduced [100]. Thus, decreased intracellular K + concentration is a common activation mode of NLRP3 inflammasome [112]. ...
Background
Neurodegenerative diseases are a common group of neurological disorders characterized by progressive loss of neuronal structure and function leading to cognitive impairment. Recent studies have shown that neuronal pyroptosis mediated by the NLRP3 inflammasome plays a crucial role in the pathogenesis of neurodegenerative diseases.
Objective and method
The NLRP3 inflammasome is a multiprotein complex that, when activated within cells, triggers an inflammatory response, ultimately leading to pyroptotic cell death of neurons. Pyroptosis is a typical pro-inflammatory programmed cell death process occurring downstream of NLRP3 inflammasome activation, characterized by the formation of pores on the cell membrane by the GSDMD protein, leading to cell lysis and the release of inflammatory factors. It has been found that NLRP3 inflammasome-mediated neuronal pyroptosis is closely associated with the development of various neurodegenerative diseases, such as Alzheimer's disease, traumatic brain injury, and Parkinson's disease. Therefore, inhibiting NLRP3 inflammasome activation and attenuating neuronal pyroptosis could potentially serve as novel strategies for the treatment of neurodegenerative diseases.
Results
The aim of this review is to explore the role of NLRP3 activation-mediated neuronal pyroptosis and neuroinflammation in neurodegenerative diseases. Firstly, we extensively discuss the relationship between NLRP3 inflammasome-mediated neuronal pyroptosis and neuroinflammation in various neurodegenerative diseases. Subsequently, we further explore the mechanisms driving NLRP3 activation and assembly, as well as the post-translational modifications regulating NLRP3 inflammasome activation.
Conclusion
Understanding these mechanisms will contribute to a deeper understanding of the link between neuronal pyroptosis and neurodegenerative diseases, and hold significant implications for the treatment and prevention of neurodegenerative diseases.
... NLRP3 inflammasome, a multiprotein complex, is mainly composed of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (6,7). It is capable of being activated by pathogen-associated molecular patterns or damage-associated molecular patterns, including virus, fungi, ATP, monosodium urate (MSU), silica crystals, fibrillar amyloid-β peptide, and aluminum salt crystals (8)(9)(10)(11)(12)(13). NLRP3 inflammasome activation regulates maturation and secretion of pro-inflammatory cytokines (such as interleukin 1β [IL-1β]), and as well as cleavage of gasdermin D (GSDMD), which promotes a lytic form of cell death called pyroptosis (14,15). ...
The NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections. However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host. Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation. We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated. Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation. Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.
... 28 Activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome by C. albicans hyphae has been proposed as the molecular mechanism discriminating between fungal colonization and disease. 29 In this regard, mice deficient in NLRP3 are more susceptible to both disseminated 30,31 and mucosal candidiasis. 32 Similarly, galactosaminogalactan exposed in the cell wall of germinated A. fumigatus drives the activation of the NLRP3 inflammasome. ...
Fungal infections present a significant global public health concern, impacting over one billion individuals worldwide and resulting in more than 3 million deaths annually. Despite considerable progress in recent years, the management of fungal infections remains challenging. The limited development of novel diagnostic and therapeutic approaches is largely attributed to our incomplete understanding of the pathogenetic mechanisms involved in these diseases. Recent research has highlighted the pivotal role of cellular metabolism in regulating the interaction between fungi and their hosts. In response to fungal infection, immune cells undergo complex metabolic adjustments to meet the energy demands necessary for an effective immune response. A comprehensive understanding of the metabolic circuits governing antifungal immunity, combined with the integration of individual host traits, holds the potential to inform novel medical interventions for fungal infections. This review explores recent insights into the immunometabolic regulation of host-fungal interactions and the infection outcome and discusses how the metabolic repurposing of immune cell function could be exploited in innovative and personalized therapeutic approaches.
... Dysbiosis of fungal microbiota sets an environment of immune dysregulation, damages the macrophages using NOD-like receptor protein 3 (NLRP3) inflammasomedependent IL-1β production, which is key antifungal immunity pathway [84,85], destruct gut epithelial lining by candidalysin, which is a fungal toxin peptide, [68] and phospholipase, which is a hydrolytic enzyme, increases biofilm formation from interaction of C. tropicalis with S. marcescens and E. coli [86], promotes hyphal formation in hosts with underlying genetic predisposition and increases intestinal permeability by inducing mucin-degrading bacteria including Akkermansia muciniphila and Ruminococcus gnavus [87]. ...
Inflammatory bowel disease (IBD) is an immune mediated chronic inflammatory disorder of gastrointestinal tract, which has underlying multifactorial pathogenic determinants such as environmental factors, susceptibility genes, gut microbial dysbiosis and a dysregulated immune response. Human gut is a frequent inhabitant of complex microbial ecosystem encompassing bacteria, viruses, parasites, fungi and other microorganisms that have an undisputable role in maintaining balanced homeostasis. All of these microbes interact with immune system and affect human gut physiology either directly or indirectly with interaction of each other. Intestinal fungi represent a smaller but crucial component of the human gut microbiome. Besides interaction with bacteriome and virome, it helps in balancing homoeostasis between pathophysiological and physiological processes, which is often dysregulated in patients with IBD. Understanding of gut mycobiome and its clinical implications are still in in its infancy as opposed to bacterial component of gut microbiome, which is more often focused. Modulation of gut mycobiome represents a novel and promising strategy in the management of patients with IBD. Emerging mycobiome-based therapies such as diet interventions, fecal microbiota transplantation (FMT), probiotics (both fungal and bacterial strains) and antifungals exhibit substantial effects in calibrating the gut mycobiome and restoring dysbalanced immune homeostasis by restoring the core gut mycobiome. In this review, we summarized compositional and functional diversity of the gut mycobiome in healthy individuals and patients with IBD, gut mycobiome dysbiosis in patients with IBD, host immune-fungal interactions and therapeutic role of modulation of intestinal fungi in patients with IBD.
... The three molecular mechanisms of NLRP3 inflammasome activation include increased ROS generation, lysosome destruction, and elevated K + efflux [13]. Studies have found [31,32] that Syk is an upstream kinase that activates the NLRP3 inflammasome. In addition, studies [15] have shown that NADPH oxidase-induced ROS is critical for NLRP3 inflammasome activation in macrophages, and ROS production has also been found to be mediated through Syk. ...
Background:
Neuronal injury in chronic cerebral hypoperfusion (CCH) is the main pathogenic factor of vascular dementia (VD). Clinically, there isn't a drug specifically for VD; instead, the majority of medications used to treat Alzheimer's disease (AD) are also used to treat VD. Based on the proven anti-inflammatory and antioxidant effects of Probucol, we hypothesized that it may have therapeutic effects on VD, but more research is required to determine its exact mechanism of action.
Methods:
In vivo experiment: We used SD rats and most commonly used bilateral carotid artery occlusion (2-VO) in VD for modeling. After successful modeling, SD rats were given Probucol 3.5 mg/kg/day for 8 weeks to evaluate the therapeutic effect. In vitro experiment: BV-2 microglia of rats were cultured and divided into Control group and Probucol group. Each group was treated with hypoxia-hypoglycemia, hypoxia-hypoglycemia hydrogen peroxide and hypoxia-hypoglycemia hydrogen peroxide Syk inhibitor respectively.
Results:
The results of immunofluorescence and Western blot showed that Probucol could significantly improve the cognitive impairment induced by CCH, and the neuronal damage was also attenuated. On the one hand, the underlying mechanism of Probucol was to reduce oxidative stress and cell apoptosis of hippocampal neurons by inhibiting the expression of phosphorylated spleen tyrosine kinase (P-Syk); On the other hand, it exerted a protective effect by reducing NLRP3-dependent cell pyroptosis and inhibiting neuroinflammation induced by microglia activation.
Conclusion:
Probucol could reduce oxidative stress and cell apoptosis by inhibiting the Syk/ROS signaling pathway, thereby improving CCH-induced cognitive impairment in vitro and in vivo.
... NLRP3 inflammasome plays a crucial role in host immune defenses against bacterial, fungal, and viral infections [43][44][45]. In the neuroinflammaging context, a typical dysregulation of TLR-mediated NLRP3 inflammasome activation within the CNS frequently occurs [41], thus contributing to increase the risk of developing several age-related and neurodegenerative diseases [46,47]. ...
... This is followed by the activation of CARD9-BCL10-MALT1 (CBM) complexdependent nuclear factor-κB (NF-κB) and the mitogen-activated protein kinase (MAPK) signaling pathways, and subsequent release of proinflammatory cytokines and chemokines, including interleukin-6 (IL-6), IL-1β, IL-23A, and CXCL1. Th17 cell polarization and the subsequent release of IL-17A are then induced, which plays a vital role in neutrophil recruitment for fungal clearance [13][14][15][16][17]. However, the excessive production of proinflammatory cytokine, such as IL-6, IL-1β and TNF-α, may trigger an inflammatory storm and induce organ damage [18]. ...
Fungal infections have emerged as a major concern among immunocompromised patients, causing approximately 2 million deaths each year worldwide. However, the regulatory mechanisms underlying antifungal immunity remain elusive and require further investigation. The E3 ligase Trim26 belongs to the tripartite motif (Trim) protein family, which is involved in various biological processes, including cell proliferation, antiviral innate immunity, and inflammatory responses. Herein, we report that Trim26 exerts protective antifungal immune functions after fungal infection. Trim26 -deficient mice are more susceptible to fungemia than their wild-type counterparts. Mechanistically, Trim26 restricts inflammatory neutrophils infiltration and limits proinflammatory cytokine production, which can attenuate kidney fungal load and renal damage during Candida infection. Trim26-deficient neutrophils showed higher proinflammatory cytokine expression and impaired fungicidal activity. We further demonstrated that excessive neutrophils infiltration in the kidney was because of the increased production of chemokines CXCL1 and CXCL2, which are mainly synthesized in the macrophages or dendritic cells of Trim26 -deficient mice after Candida albicans infections. Together, our study findings unraveled the vital role of Trim26 in regulating antifungal immunity through the regulation of inflammatory neutrophils infiltration and proinflammatory cytokine and chemokine expression during candidiasis.
... R. Taylor et al., 2007;Whitney et al., 2014) and TLR genes (Bochud et al., 2008;Carvalho et al., 2008;Netea et al., 2002). Although fungal NLR ligands are not fully defined yet, the NLRP3 inflammasome process has been shown to influence the defense against mycobiota (Gross et al., 2009). PRRs on immune cells initiate downstream intracellular events that eventually lead to the clearance or tolerance of fungal cells within the body (Figure 3b). ...
Fungi are the cause of more than a billion infections in humans every year, although their interactions with the host are still neglected compared to bacteria. Major systemic fungal infections are very unusual in the healthy population, due to the long history of coevolution with the human host. Humans are routinely exposed to environmental fungi and can host a commensal mycobiota, which is increasingly considered as a key player in health and disease. Here, we review the current knowledge on host‐fungi coevolution and the factors that regulate their interaction. On one hand, fungi have learned to survive and inhabit the host organisms as a natural ecosystem, on the other hand, the host immune system finely tunes the response toward fungi. In turn, recognition of fungi as commensals or pathogens regulates the host immune balance in health and disease. In the human gut ecosystem, yeasts provide a fingerprint of the transient microbiota. Their status as passengers or colonizers is related to the integrity of the gut barrier and the risk of multiple disorders. Thus, the study of this less known component of the microbiota could unravel the rules of the transition from passengers to colonizers and invaders, as well as their dependence on the innate component of the host's immune response.
This article is categorized under: Infectious Diseases > Environmental Factors
Immune System Diseases > Environmental Factors
Infectious Diseases > Molecular and Cellular Physiology
... In the second model, matrix proteins, through integrins, directly activate kinases (e.g. FAK, SYK, Pyk (Chung et al, 2016;Gross et al, 2009;Hara et al, 2013;Lin et al, 2015)) or GTPases such as RhoA (Gao et al, 2016;Xu et al, 2014) that are involved in posttranslational control of inflammasome activation, e.g. NLRP3 and Pyrin, respectively. ...
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.
... The activation of the inflammasome is mediated by a nuclear factor kappa-light-chain-enhancer of an activated B cell (NF-κB) [116]. The NLRP3 inflammasome plays a significant role in mediating the host immune defense against several bacterial, fungal, and viral infections [117][118][119]. The uncontrolled activation of the NLRP3 inflammasome contributes towards the pathogenesis of several diseases, including autoimmune diseases, diabetes, gout, cryopyrin-associated periodic syndromes, and atherosclerosis [113,120]. ...
Ischemic thrombotic disease, characterized by the formation of obstructive blood clots within arteries or veins, is a condition associated with life-threatening events, such as stroke, myocardial infarction, deep vein thrombosis, and pulmonary embolism. The conventional therapeutic strategy relies on treatments with anticoagulants that unfortunately pose an inherent risk of bleeding complications. These anticoagulants primarily target clotting factors, often overlooking upstream events, including the release of neutrophil extracellular traps (NETs). Neutrophils are integral components of the innate immune system, traditionally known for their role in combating pathogens through NET formation. Emerging evidence has now revealed that NETs contribute to a prothrombotic milieu by promoting platelet activation, increasing thrombin generation, and providing a scaffold for clot formation. Additionally, NET components enhance clot stability and resistance to fibrinolysis. Clinical and preclinical studies have underscored the mechanistic involvement of NETs in the pathogenesis of thrombotic complications, since the clots obtained from patients and experimental models consistently exhibit the presence of NETs. Given these insights, the inhibition of NETs or NET formation is emerging as a promising therapeutic approach for ischemic thrombotic diseases. Recent investigations also implicate a role for the nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome as a mediator of NETosis and thrombosis, suggesting that NLRP3 inhibition may also hold potential for mitigating thrombotic events. Therefore, future preclinical and clinical studies aimed at identifying and validating NLRP3 inhibition as a novel therapeutic intervention for thrombotic disorders are imperative.
... To investigate the precise mechanism of MANF on SIC injury, the relationship between MANF and cell pyroptosis was further determined. NLRP3 was the crucial mediator of cell pyroptosis (20,21). Therefore, cell pyroptosis was determined after suppressed NLRP3 and knockdown of MANF in the SIC in vitro model. ...
Background
Sepsis-induced cardiomyopathy (SIC) is a common complication of sepsis accompanied by high prevalence and mortality in sepsis patients. Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neurotrophic factor, and it exerts critical functions in various diseases, including heart diseases, while its effect on SIC remains elusive. Hence, we aimed to investigate the action of MANF on SIC.
Methods
This study was under the guidance of Gongli Hospital, Shanghai, China from January 2021 to December 2021. H9c2 cells and mice were induced by LPS to establish SIC in vitro and in vivo models. qRT-PCR and Western blot were used to determine gene and protein expressions. The levels of MANF, Interleukin-1β (IL-1β), Interleukin 18 (IL-18), creatine kinase-MB (CK-MB), and cardiac troponin I (cTn I) were detected using ELISA assay. Cell pyroptosis determination was performed by flow cytometry. The DCFDA assay kit was used to determine ROS production.
Results
In SIC in vitro model, LPS induced cell pyroptosis (P<0.001) and ROS accumulation (P<0.001). Besides, MANF was decreased in LPS-induced H9c2 cells (P<0.001) and SIC patients (P<0.001). In addition, overexpression of MANF ameliorated SIC-induced injury in H9C2 cells (P<0.001). Furthermore, inhibition of NLRP3 rescued the function of MANF on SIC-induced injury in H9C2 cells (P<0.001). Moreover, enforced MANF suppressed the SIC-induced injury in vivo model (P<0.001).
Conclusion
MANF was down-regulated in SIC. Overexpressed MANF ameliorated the SIC injury by inhibiting NLRP3-mediated pyroptosis.
... Although activated by different types of PAMPs and DAMPs, they have similar downstream effector functions, including the activation of GSDMD-mediated pyroptosis and the activation of caspase-1, leading to subsequent proteolytic maturation of IL-1β and IL-18 and their secretion through GSDMD-mediated pores (23)(24)(25)(26). Among the canonical inflammasomes, NLRP3 is the most studied, being largely involved in both innate and adaptive immunity after activation by either whole pathogens, including fungi, bacteria, and viruses, or by host-derived molecules, such as fibrillar amyloid-β (Aβ) peptide, as well as extracellular ATP and glucose (27)(28)(29)(30)(31). ...
Kidney transplantation is the best available renal replacement therapy for patients with end-stage kidney disease and is associated with better quality of life and patient survival compared with dialysis. However, despite the significant technical and pharmaceutical advances in this field, kidney transplant recipients are still characterized by reduced long-term graft survival. In fact, almost half of the patients lose their allograft after 15–20 years. Most of the conditions leading to graft loss are triggered by the activation of a large immune-inflammatory machinery. In this context, several inflammatory markers have been identified, and the deregulation of the inflammasome (NLRP3, NLRP1, NLRC4, AIM2), a multiprotein complex activated by either whole pathogens (including fungi, bacteria, and viruses) or host-derived molecules, seems to play a pivotal pathogenetic role. However, the biological mechanisms leading to inflammasome activation in patients developing post-transplant complications (including, ischemia-reperfusion injury, rejections, infections) are still largely unrecognized, and only a few research reports, reviewed in this manuscript, have addressed the association between abnormal activation of this pathway and the onset/development of major clinical effects. Finally, the regulation of the inflammasome machinery could represent in future a valuable therapeutic target in kidney transplantation.
... Phagocytosis of IgG-opsonized latex beads activates TFEB in macrophage-like RAW264.7 cells and primary BMDMs via FcγR and Syk (18). Syk is also a critical component of C-type lectin receptor signaling, linking Dectin-1 to Card9 (29). In response to A. fumigatus, Dectin-1 and Card9 are required for optimal activation of TFEB and TFE3, suggesting that Syk may also be involved in this context. ...
Myeloid phagocytes of the respiratory immune system, such as neutrophils, monocytes, and alveolar macrophages, are essential for immunity to Aspergillus fumigatus , the most common etiologic agent of mold pneumonia worldwide. Following the engulfment of A. fumigatus conidia, fusion of the phagosome with the lysosome is a critical process for killing conidia. TFEB and TFE3 are transcription factors that regulate lysosomal biogenesis under stress and are activated by inflammatory stimuli in macrophages, but it is unknown whether TFEB and TFE3 contribute to anti- Aspergillus immunity during infection. We found that lung neutrophils express TFEB and TFE3, and their target genes were upregulated during A. fumigatus lung infection. In addition, A. fumigatus infection induced nuclear accumulation of TFEB and TFE3 in macrophages in a process regulated by Dectin-1 and CARD9. Genetic deletion of Tfeb and Tfe3 impaired macrophage killing of A. fumigatus conidia. However, in a murine immune-competent Aspergillus infection model with genetic deficiency of Tfeb and Tfe3 in hematopoietic cells, we surprisingly found that lung myeloid phagocytes had no defects in conidial phagocytosis or killing. Loss of TFEB and TFE3 did not impact murine survival or clearance of A. fumigatus from the lungs. Our findings indicate that myeloid phagocytes activate TFEB and TFE3 in response to A. fumigatus , and while this pathway promotes macrophage fungicidal activity in vitro , genetic loss can be functionally compensated in the lung, resulting in no measurable defect in fungal control and host survival.
... 7 As a key trigger of inflammatory diseases, IL-1β is a central coordinator of immune responses to various classes of pathogens. 13 IL-1β agents have demonstrated significant efficacy in the clinical treatment of cryopyrin-related auto-inflammatory syndrome, and subsequent clinical trials of other NLRP3associated diseases have also been carried out. 14,15 However, there are concerns about the current treatment modalities. ...
Background
Despite all modern advances in medicine, an effective drug for treating sepsis has yet to be found. The discovery of CMPK2 spurred hopes for the treatment of sepsis. However, CMPK2‐untapped target inhibitors are still an enormous obstacle that has hindered the CMPK2‐centric treatment of sepsis.
Methods
Here, we found that the CMPK2 gene is highly expressed in the whole blood of sepsis patients by RNA‐Seq. First, recombinant CMPK2 was purified by a eukaryotic expression purification system, and the activity of recombinant CMPK2 was detected by the ADP‐GLO assay. Second, we developed an affinity MS strategy combined with quantitative lysine reactivity profiling to discover CMPK2 ligands from the active ingredients of Chinese herbs. In addition, the dissociation constant K d of the ligand and the target protein CMPK2 was further detected by microscale thermophoresis technology. Third, we used this strategy to identify a naturally sourced small molecule, dracorhodin (DP). Using mass spectrometry‐based quantitative lysine reactivity profiling combined with a series of mutant tests, the results show that K265 acts as a bright hotspot of DP inhibition of CMPK2. Fourth, immune‐histochemical staining, ELISAs, RT‐qPCR, flow cytometry and immunoblotting were used to illustrate the potential function and related mechanism of DP in regulating sepsis injury.
Results
Our results suggest that DP exerts powerful anti‐inflammatory effects by regulating the NLRP3 inflammasome via the lipopolysaccharide (LPS)‐induced CMPK2 pathway. Strikingly, DP significantly attenuated LPS‐induced sepsis in a mouse model, but its effect was weakened in mice with myeloid‐specific Cmpk2 ablation.
Conclusion
We provide a new framework that provides more valuable information for new therapeutic approaches to sepsis, including the establishment of screening strategies and the development of target drugs to provide a theoretical basis for ultimately improving clinical outcomes for sepsis patients. Collectively, these findings reveal that DP is a promising CMPK2 inhibitor for the treatment of sepsis.
... Several inflammasomes, including NLRP1, NLRP2, NLRP3, AIM2, and NLRC4, have been discovered [27]. The immune receptor protein NLRP3, adaptor protein ASC (apoptosis associated speck-like protein containing a caspase recruitment domain), and inflammatory protease caspase-1 make up the most extensively studied inflammasome, which reacts to microbial infections, endogenous danger signals, metabolic risk factors, and environmental stimuli [28,29]. An earlier investigation revealed that the gene expression of NLRP3 was so low that it was insufficient to activate the assembly of an inflammasome during resting conditions. ...
Pyroptosis is a type of regulated cell death that relies on caspases, vesicles, and the cleavage of gasdermin proteins (which create pores in the cell membrane). The nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, which is involved in this process, is the most widely studied inflammasome. Caspase-1 activates pro-inflammatory cytokines, such as IL-1β and IL-18. Gasdermin D (GSDMD) is the most important executive protein. GSDMD, a substrate rather than an upstream protease, determines the occurrence of pyroptosis. Pyroptosis is essential for maintaining body homeostasis, but excessive or poorly regulated cell death can aggravate the inflammatory response. Undoubtedly, this will be an important direction for future research on Alzheimer’s disease (AD). Here, we review recent research progress on the morphological characteristics, molecular mechanisms, and role of pyroptosis in the context of AD, thereby providing new directions for identifying potential disease biomarkers and treatment strategies for AD.
... The NLRP3 inflammasome is among the most studied, and is shown to be of critical importance for host immune defense against bacterial, fungal, and viral infections (10)(11)(12)(13)(14). In macrophages, NLRP3 inflammasome formation is a two-step process. ...
Neutrophil recruitment to sites of infection and inflammation is an essential process in the early innate immune response. Upon activation, a subset of neutrophils rapidly assembles the multiprotein complex known as the NLRP3 inflammasome. The NLRP3 inflammasome forms at the microtubule organizing center, which promotes the formation of interleukin (IL)-1β and IL-18, essential cytokines in the immune response. We recently showed that mice deficient in NLRP3 (NLRP3-/-) have reduced neutrophil recruitment to the peritoneum in a model of thioglycolate-induced peritonitis. Here, we tested the hypothesis that this diminished recruitment could be, in part, the result of defects in neutrophil chemotaxis. We find that NLRP3-/- neutrophils show loss of cell polarization, as well as reduced directionality and velocity of migration toward increasing concentrations of leukotriene B4 (LTB4) in a chemotaxis assay in vitro, which was confirmed through intravital microscopy of neutrophil migration toward a laser-induced burn injury of the liver. Furthermore, pharmacologically blocking NLRP3 inflammasome assembly with MCC950 in vitro reduced directionality but preserved nondirectional movement, indicating that inflammasome assembly is specifically required for polarization and directional chemotaxis, but not cell motility per se. In support of this, pharmacological breakdown of the microtubule cytoskeleton via nocodazole treatment induced cell polarization and restored nondirectional cell migration in NLRP3-deficient neutrophils in the LTB4 gradient. Therefore, NLRP3 inflammasome assembly is required for establishment of cell polarity to guide the directional chemotactic migration of neutrophils.
... Wide ranges of stimuli that originate from pathogenic, endogenic, or environmental resources can induce NLRP3 activation and response. Some of the pathogens that stimulate NLRP3 include Staphylococcus aureus, Listeria monocytogenes (Mariathasan et al., 2006), Candida albicans (Gross et al., 2009), and the influenza A virus (Kanneganti et al., 2006). Also, as mentioned, some endogen stimuli, such as extracellular adenosine triphosphate (ATP), hyaluronan, amyloid-β fibrils, and uric acid crystals, can stimulate NLRP3 as well (Cassel et al., 2008;Cruz et al., 2007;Rashidi et al., 2020). ...
Psoriasis is an inflammatory skin disease whose pathophysiology is attributed to both innate and adaptive immune cells and molecules. Despite the crucial roles of the immune system in psoriasis, it cannot be categorized as an autoimmune disease because of the lack of main signs of autoimmunity, such as specific antibodies, well-defined antigens, and autoimmune genetic risk factors. The presence of some cellular and molecular properties, such as the presence of neutrophils in skin lesions and the activation of the innate immune system, attributes psoriasis to a group of diseases called autoinflammatory disorders. Autoinflammatory diseases refer to a group of inherited disorders whose main manifestations are recurrent fever, a high level of acute-phase reactant, and a tendency for inflammation of the skin, joints, and other organs like the nervous system. In most autoinflammatory disorders, it has been seen that complexes of the high-molecular-weight protein named inflammasomes have significant roles. The inflammasome complex usually is formed and activated in the stimulated immune cell cytoplasm, and its activation consequently leads to inflammatory events such as producing of active caspase-1, mature interleukin-1β (IL-1β), and IL-18 and can cause an inflammatory programmed cell death called pyroptosis. Since the identification of inflammasomes, it has been shown that there are close links between them and hereditary and acquired autoinflammatory diseases like psoriasis. In this review, we aim to focus on well-defined inflammasome and their role in the pathophysiology of psoriasis.
... Although some studies have shown that ASC is required for the activation of the NLRP1 inflammasome [28,29], others have indicated the possibility of NLRP1 to directly recruit pro-caspase 1 [30] and caspase-5 [5], via a CARD-CARD domain interaction. One of the most characterized inflammasome of NLR family is NLRP3 inflammasome, crucially involved in host immune defences against bacterial, fungal and viral infections [10, [31][32][33][34]. At present, a two-signal model has been proposed for NLRP3 inflammasome activation: a first or priming signal, provided by microbial components or endogenous cytokines, and a second or activating signal, consisting of extracellular adenosine triphosphate (ATP), pore-forming toxins, particulate matter, or multiple molecular and cellular events (i.e., mitochondrial dysfunction, the production of excessive reactive oxygen species (ROS), lysosomal damage, etc.) [10]. ...
Neuroinflammation plays a crucial role in the onset and the progression of several neuropathologies, from neurodegenerative disorders to migraine, from Rett syndrome to post-COVID 19 neurological manifestations. Inflammasomes are cytosolic multiprotein complexes of the innate immune system that fuel inflammation. They have been under study for the last twenty years and more recently their involvement in neuro-related conditions has been of great interest as possible therapeutic target. The role of oxidative stress in inflammasome activation has been described, however the exact way of action of specific endogenous and exogenous oxidants needs to be better clarified. In this review, we provide the current knowledge on the involvement of inflammasome in the main neuropathologies, emphasizing the importance to further clarify the role of oxidative stress in its activation including the role of mitochondria in inflammasome-induced neuroinflammation.
... Upon C. albicans or zymosan exposure, Syk activation induces ROS generation via NOX (17,53,54). ...
Recognition of pathogen-associated molecular patterns can trigger the IRE1α arm of the endoplasmic reticulum (ER) stress response in innate immune cells. This process maintains ER homeostasis and also coordinates diverse immunomodulatory programs during bacterial and viral infections. However, the role of innate IRE1α signaling in response to fungal pathogens remains elusive. Here, we report that systemic infection with the human opportunistic fungal pathogen Candida albicans induces proinflammatory IRE1α hyperactivation in myeloid cells that leads to fatal kidney immunopathology. Mechanistically, simultaneous activation of the TLR/IL-1R adaptor protein MyD88 and the C-type lectin receptor Dectin-1 by C. albicans induced NADPH oxidase-driven generation of reactive oxygen species that caused ER stress and IRE1α-dependent overexpression of key inflammatory mediators such as IL-1β, IL-6, CCL5, PGE2 and TNFα. Selective ablation of IRE1α in leukocytes, or treatment with an IRE1α pharmacological inhibitor, mitigated kidney inflammation and prolonged the survival of mice with systemic C. albicans infection. Therefore, controlling IRE1α hyperactivation may be useful for impeding the immunopathogenic progression of disseminated candidiasis.
... Emerging evidence indicates that the NLRP3 inflammasome plays an indispensable role in the defense response to Candida infection. Studies have shown that NLRP3-deficient mice infected with C. albicans display lower serum IL-1β levels, reduced survival, higher fungal burdens, and higher clinical severity scores than wild-type mice [31,32]. Our results indicate that C. albicans activates the NLRP3 signal pathway. ...
Background
It is well recognized that both smoke and Candida infection are crucial risk factors for oral mucosal diseases. The nucleotide-binding domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and its downstream effectors, interleukin (IL)-1β and IL-18, are pivotal to the host defense against Candida and other pathogens.
Methods
The present study was designed to explore the effects of cigarette smoke and C. albicans on the NLRP3 inflammasome and its downstream signal pathway via in vitro cell model. Oral epithelial cells (Leuk-1 cells) were exposed to cigarette smoke extract (CSE) for 3 days and/or challenged with C. albicans.
Results
Microscopically, Leuk-1 cells exerted a defense response to C. albicans by markedly limiting the formation of germ tubes and microcolonies. CSE clearly eliminated the defense response of Leuk-1 cells. Functionally, CSE repressed NLRP3 inflammasome, and IL-1β and IL-18 activation induced by C. albicans in Leuk-1 cells.
Conclusion
Our results suggested that in oral epithelial cells, the NLRP3 inflammasome might be one of the target pathways by which CSE attenuates innate immunity and leads to oral disorders.
... The Nod-like receptor protein 3 (NLRP3) inflammasome is an intracellular multiprotein complex structure that the host immune system utilizes to fight infections against bacteria, fungi, and viruses. (Allen et al., 2009;Gross et al., 2009;Kanneganti et al., 2006;Thomas et al., 2009). Overexpression of NLRP3 has been detected in monocytes/macrophages, antigen-presenting cells like dendritic cells, and neutrophils (Jha et al., 2017;Kummer et al., 2007;Sutterwala et al., 2006). ...
When used as an alternative source of drugs to treat inflammation-associated diseases, phytochemicals with anti-inflammatory properties provide beneficial impacts. Galangin is one of the most naturally occurring flavonoids. Galangin has many biological activities, such as anti-inflammatory, antioxidant, antiproliferative, antimicrobial, anti-obesity, antidiabetic, and anti-genotoxic activities. We observed that galangin was well tolerated and positively impacted disease underlying inflammation for the renal, hepatic, central nervous system, cardiovascular, gastrointestinal system, skin, and respiratory disorders, as well as ulcerative colitis, acute pancreatitis, retinopathy, osteoarthritis, osteoporosis, and rheumatoid arthritis. Galangin anti-inflammatory effects are mediated mainly by suppressing p38 mitogen-activated protein kinases, nuclear factor-kappa B, and nod-like receptor protein 3 signals. These effects are confirmed and supported by molecular docking. Clinical translational research is required to accelerate the bench-to-bedside transfer and determine whether galangin can be utilised as a safe, natural source of pharmaceutical anti-inflammatory medication for humans.
Cognitive impairment is common in peripheral diseases such as chronic kidney disease (CKD). Kidney transplantation reverses cognitive impairment, indicating that cognitive impairment driven by CKD is therapeutically amendable. Yet, we lack mechanistic insights allowing targeted therapies. Using a combination of mouse models (including mice with neuron-specific IL-1R1 deficiency), single cell analyses (single nuclei RNA sequencing and single cell thallium automethallography), human samples and in vitro experiments we demonstrate that microglia activation impairs neuronal potassium homeostasis and impairs cognition in CKD. CKD conditions disrupt the barrier of brain endothelial cells in vitro and the blood-brain barrier in vivo, establishing that brain cells are exposed to uremic conditions. Exposure to uremic conditions impairs calcium homeostasis in microglia, enhances microglial potassium (K⁺) efflux via the calcium-dependent channel KCa3.1, and induces p38-MAPK associated IL-1β maturation in microglia. Restoring K⁺ homeostasis in microglia using a KCa3.1-specific inhibitor (TRAM34) improves CKD-triggered cognitive impairment. Likewise, inhibition of the IL-1β receptor 1 (IL-1R1) using anakinra or genetically abolishing neuronal IL-1R1 expression in neurons prevent CKD-mediated reduced neuronal potassium turnover and CKD-induced impaired cognition. Thus, in CKD mice impaired cognition can be ameliorated by either preventing microglia activation or inhibiting IL-1R-signaling in neurons. These data suggest that potassium efflux from microglia triggers their activation, which promotes microglia IL-1β release and IL-1R1-mediated neuronal dysfunction in CKD. This study provides new mechanistic insight into cognitive impairment in association with CKD and identifies possible new therapeutic approaches.
A significant increase in the incidence of Candida -mediated infections has been observed in the last decade, mainly due to rising numbers of susceptible individuals. Recently, the World Health Organization published its first fungal pathogen priority list, with Candida species listed in medium, high, and critical priority categories. This review is a synthesis of information and recent advances in our understanding of two of these species —Candida albicans and Candida glabrata . Of these, C. albicans is the most common cause of candidemia around the world and is categorized as a critical priority pathogen. C. glabrata is considered a high-priority pathogen and has become an increasingly important cause of candidemia in recent years. It is now the second most common causative agent of candidemia in many geographical regions. Despite their differences and phylogenetic divergence, they are successful as pathogens and commensals of humans. Both species can cause a broad variety of infections, ranging from superficial to potentially lethal systemic infections. While they share similarities in certain infection strategies, including tissue adhesion and invasion, they differ significantly in key aspects of their biology, interaction with immune cells, host damage strategies, and metabolic adaptations. Here we provide insights on key aspects of their biology, epidemiology, commensal and pathogenic lifestyles, interactions with the immune system, and antifungal resistance.
Inflammation triggers specific metabolic pathways and if not resolved, translates into several painful diseases such as rheumatoid arthritis, lupus, Alzheimer's disease, cardiovascular disorders and psoriasis. Various processes have been explored to understand the factors behind inflammation and consequently, many mechanisms have been examined to suppress it. The nucleotide-binding domain like receptor 3 (NLRP3) inflammasome is an example of such factors which is responsible for triggering sterile and microbe induced inflammation. Studies of genetic variants of the related gene have revealed insights into the mRNA expression pathways that may help researchers to identify crucial disease mechanisms.
This book is a review of the scientific findings of distinguished scholars who have studied NLRP3 inflammasome activation and its contribution in worsening the outcomes of inflammatory disorders.
This collection of chapters covers many aspects of the multifaceted role of NLRP3 inflammasome. Beginning with airway inflammation and fibrosis, it progresses to explore its involvement in pulmonary hypertension, heart diseases, tuberculosis, cardiovascular complications, and childhood asthma. Additionally, it examines the inflammasome's impact on protozoan parasitic infections and neuropathic pain. The chapters not only elucidate the intricate mechanisms of NLRP3 activation but also discuss potential inhibitors and therapeutic targets. Readers will gain a comprehensive understanding of the NLRP3 inflammasome's diverse implications across different physiological contexts. The book includes references making this book a valuable treatise of insights for researchers, clinicians, and healthcare professionals.
Extracellular proteases are key factors contributing to the virulence of pathogenic fungi from the genus Candida. Their proteolytic activities are crucial for extracting nutrients from the external environment, degrading host defenses, and destabilizing the internal balance of the human organism. Currently, the enzymes most frequently described in this context are secreted aspartic proteases (Saps). This review comprehensively explores the multifaceted roles of Saps, highlighting their importance in biofilm formation, tissue invasion through the degradation of extracellular matrix proteins and components of the coagulation cascade, modulation of host immune responses via impairment of neutrophil and monocyte/macrophage functions, and their contribution to antifungal resistance. Additionally, the diagnostic challenges associated with Candida infections and the potential of Saps as biomarkers were discussed. Furthermore, we examined the prospects of developing vaccines based on Saps and the use of protease inhibitors as adjunctive therapies for candidiasis. Given the complex biology of Saps and their central role in Candida pathogenicity, a multidisciplinary approach may pave the way for innovative diagnostic strategies and open new opportunities for innovative clinical interventions against candidiasis.
Parasitic diseases are a serious global health concern, causing many common and severe infections, including Chagas disease, leishmaniasis,and schistosomiasis. The NLRP3 inflammasome belongs to the NLR (nucleotide-binding domain leucine-rich-repeat-containing proteins) family, which are cytosolic proteins playing key roles in the detection of pathogens. NLRP3 inflammasomes are activated in immune responses to Plasmodium,Leishmania, Toxoplasma gondii, Entamoeba histolytica,Trypanosoma cruzi and other parasites. The role of NLRP3 is not fully understood, but it is a crucial component of the innate immune response to parasitic infections and its functions as a sensor triggering the inflammatory response to the invasive parasites. However, while this response can limit the parasites’ growth, it can also result in potentially catastrophic host pathology. This makes it essential to understand how NLRP3 interacts with parasites to initiate the inflammatory response. Plasmodium hemozoin, Leishmania glycoconjugate lipophosphoglycan (LPG) and E. histolytica Gal/GalNAc lectin can stimulate NLRP3 activation, while the dense granule protein 9 (GRA9) of T. gondii has been shown to suppress it. Several other parasitic products also have diverse effects on NLRP3 activation. Understanding the mechanism of NLRP3 interaction with these products will help to develop advanced therapeutic approaches to treat parasitic diseases. This review summarizes current knowledge of the NLRP3 inflammasome’s action on the immune response to parasitic infections and aims to determine the mechanisms through which parasitic molecules either activate or inhibit its action.
The uterine cervix is one of the key factors involved in ensuring a proper track of gestation and labor. At the end of the gestational period, the cervix undergoes extensive changes, which can be summarized as a transformation from a non-favorable cervix to one that is soft and prone to dilation. During a process called cervical ripening, fundamental remodeling of the cervical extracellular matrix (ECM) occurs. The cervical ripening process is a derivative of many interlocking and mutually driving biochemical and molecular pathways under the strict control of mediators such as inflammatory cytokines, nitric oxide, prostaglandins, and reactive oxygen species. A thorough understanding of all these pathways and learning about possible triggering factors will allow us to develop new, better treatment algorithms and therapeutic goals that could protect women from both dysfunctional childbirth and premature birth. This review aims to present the possible role of the NLRP3 inflammasome in the cervical ripening process, emphasizing possible mechanisms of action and regulatory factors.
Candida albicans (C. albicans) is a prevalent opportunistic pathogen that causes mucocutaneous and systemic infections, particularly in immunocompromised individuals. Macrophages play a crucial role in eliminating C. albicans in local and bloodstream contexts, while also regulating antifungal immune responses. However, C. albicans can induce macrophage lysis through pyroptosis, a type of regulated cell death. This process can enable C. albicans to escape from immune cells and trigger the release of IL-1β and IL-18, which can impact both the host and the pathogen. Nevertheless, the mechanisms by which C. albicans triggers pyroptosis in macrophages and the key factors involved in this process remain unclear. In this review, we will explore various factors that may influence or trigger pyroptosis in macrophages induced by C. albicans, such as hypha, ergosterol, cell wall remodeling, and other virulence factors. We will also examine the possible immune response following macrophage pyroptosis.
Organ transplantation stands as a pivotal achievement in modern medicine, offering hope to individuals with end-stage organ diseases. Advancements in immunology led to improved organ transplant survival through the development of immunosuppressants, but this heightened susceptibility to fungal infections with nonspecific symptoms in recipients. This review aims to establish an intricate balance between immune responses and fungal infections in organ transplant recipients. It explores the fundamental immune mechanisms, recent advances in immune response dynamics, and strategies for immune modulation, encompassing responses to fungal infections, immunomodulatory approaches, diagnostics, treatment challenges, and management. Early diagnosis of fungal infections in transplant patients is emphasized with the understanding that innate immune responses could potentially reduce immunosuppression and promise efficient and safe immuno-modulating treatments. Advances in fungal research and genetic influences on immune-fungal interactions are underscored, as well as the potential of single-cell technologies integrated with machine learning for biomarker discovery. This review provides a snapshot of the complex interplay between immune responses and fungal infections in organ transplantation and underscores key research directions.
Background:
Paracoccidioidomycosis is the most prevalent systemic mycosis in Latin America, with a high incidence in Brazil, Colombia and Venezuela, and constitutes a serious public health problem, a frequent cause of morbidity and disability for work. Some mechanisms of cell death are described as important tools in infectious processes. When apoptosis is blocked, RIPK (Receptor-interacting protein kinase) 3 dependent, a caspase-independent form of cell death, can limit the replication and spread of pathogens. Some molecules that mediate necroptosis include RIPK3 and have been extensively studied due to their signalling mechanism and pathological function. RIPK3 activates NLRP1 and NLRP3-mediated inflammasome formation. Caspase-1 has an important role in processing the cytokines ILβ and IL18 to their active form. Such molecules are part of the inflammasome characterization, whose caspase-1-dependent activation promotes the death of pyroptotic cells and the secretion of proinflammatory cytokines. Knowledge about the mechanisms of pathogen-mediated cell death can be useful for understanding of the pathogenesis of infections and inflammatory conditions.
Objective:
The objective of this work was to identify the mechanisms of programmed cell death and inflammasome components in human oral mucosal lesions of paracoccidioidomycosis through immunohistochemical methods and identification of RIPK-3, IL1β, IL18, NLRP-1 and caspase-1. Thirty specimens were included, and a histopathological analysis of the lesions was performed using haematoxylin-eosin staining.
Results:
Our results on in situ expression of inflammasome elements and programmed cell death showed increased expression of IL-1β, NLRP-1, caspase-1 and RIPK-3. We suggest that inflammasome complex participate in the immunopathogenesis in paracoccidioidomycosis oral lesions in an interplay with RIPK3.
Inflammasomes are a group of protein complex located in cytoplasm and assemble in response to a wide variety of pathogen‐associated molecule patterns, damage‐associated molecule patterns, and cellular stress. Generally, the activation of inflammasomes will lead to maturation of proinflammatory cytokines and pyroptotic cell death, both associated with inflammatory cascade amplification. A sensor protein, an adaptor, and a procaspase protein interact through their functional domains and compose one subunit of inflammasome complex. Under physiological conditions, inflammasome functions against pathogen infection and endogenous dangers including mtROS, mtDNA, and so on, while dysregulation of its activation can lead to unwanted results. In recent years, advances have been made to clarify the mechanisms of inflammasome activation, the structural details of them and their functions (negative/positive) in multiple disease models in both animal models and human. The wide range of the stimuli makes the function of inflammasome diverse and complex. Here, we review the structure, biological functions, and therapeutic targets of inflammasomes, while highlight NLRP3, NLRC4, and AIM2 inflammasomes, which are the most well studied. In conclusion, this review focuses on the activation process, biological functions, and structure of the most well‐studied inflammasomes, summarizing and predicting approaches for disease treatment and prevention with inflammasome as a target. We aim to provide fresh insight into new solutions to the challenges in this field.
Infection by intracellular pathogens can trigger activation of the IRE1α branch of the unfolded protein response (UPR), which then modulates innate immunity and infection outcomes during bacterial or viral infection. However, the mechanisms by which infection activates IRE1α have not been fully elucidated. While recognition of microbe-associated molecular patterns can activate IRE1α, it is unclear whether this depends on the canonical role of IRE1α in detecting misfolded proteins. Here, we report that Candida albicans infection of macrophages results in IRE1α activation through C-type lectin receptor signaling, reinforcing a role for IRE1α as a central regulator of host responses to infection by a broad range of pathogens. However, IRE1α activation was not preceded by protein misfolding in response to either C. albicans infection or lipopolysaccharide treatment, implicating a non-canonical mode of IRE1α activation after recognition of microbial patterns. Investigation of the phenotypic consequences of IRE1α activation in macrophage antimicrobial responses revealed that IRE1α activity enhances the fungicidal activity of macrophages. Macrophages lacking IRE1α activity displayed inefficient phagolysosomal fusion, enabling C. albicans to evade fungal killing and escape the phagosome. Together, these data provide mechanistic insight for the non-canonical activation of IRE1α during infection, and reveal central roles for IRE1α in macrophage antifungal responses.
Human fungal pathogens are a deadly and underappreciated risk to global health that most severely affect immunocompromised individuals. A virulence attribute shared by some of the most clinically relevant fungal species is their ability to survive inside macrophages and escape from these immune cells. In this review, we discuss the mechanisms behind intracellular survival and elaborate how escape is mediated by lytic and non‐lytic pathways as well as strategies to induce programmed host cell death. We also discuss persistence as an alternative to rapid host cell exit. In the end, we address the consequences of fungal escape for the host immune response and provide future perspectives for research and development of targeted therapies.
Unabated activation of NLRP3 inflammasome activation is linked with the pathogenesis of various inflammatory disorders. PLK1 has been widely studied for its role in mitosis. Here, employing both pharmacological and genetic approaches, we demonstrated that PLK1 promoted NLRP3 inflammasome activation at cell interphase. Using an unbiased Bio-ID screen for PLK1 interactome in macrophages, we showed an enhanced proximal association of NLRP3 with PLK1 upon NLRP3 inflammasome activation. We further confirmed the interaction between PLK1 and NLRP3, and identified the interacting domains. Mechanistically, we showed that PLK1 orchestrated microtubule organizing center (MTOC) structure and NLRP3 subcellular positioning upon inflammasome activation. Treatment with a selective PLK1 kinase inhibitor suppressed IL1B production in in-vivo inflammatory models, including lipopolysaccharide-induced endotoxemia and monosodium urate-induced peritonitis in mice. Our results uncover an unprecedented role of PLK1 in regulating NLRP3 inflammasome activation during interphase, and identify pharmacological inhibition of PLK1 as a potential therapeutic strategy for inflammatory diseases with excessive NLRP3 inflammasome activation.
The NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in the innate immune system and is a three-part macromolecular complex comprising the NLRP3 protein, apoptosis-associated speck-like protein containing a CARD (ASC) and the cysteine protease pro-caspase-1. When the NLRP3 inflammasome is activated, it can produce interleukin (IL)- 1β and IL-18 and eventually lead to inflammatory cell pyroptosis. Related studies have demonstrated that the NLRP3 inflammasome can induce an immune response and is related to the occurrence and development of gynecological diseases, such as endometriosis, polycystic ovary syndrome and breast cancer. NLRP3 inflammasome inhibitors are beneficial for maintaining cellular homeostasis and tissue health and have been found effective in targeting some gynecological diseases. However, excessive inhibitor concentrations have been found to cause adverse effects. Therefore, proper control of NLRP3 inflammasome activity is critical. This paper summarizes the structure and function of the NLRP3 inflammasome and highlights the therapeutic potential of targeting it in gynecological diseases, such as endometriosis, polycystic ovary syndrome and breast cancer The application of NLRP3 inflammasome inhibitors is also discussed.
The innate immune response represents the first line of host defense, and it is able to detect pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) through a variety of pattern recognition receptors (PRRs). Among these PRRs, certain cytosolic receptors of the NLRs family (specifically NLRP1, NLRP3, NLRC4, and NAIP) or those containing at least a pyrin domain (PYD) such as pyrin and AIM2, activate the multimeric complex known as inflammasome, and its effector enzyme caspase-1. The caspase-1 induces the proteolytic maturation of the pro-inflammatory cytokines IL-1ß and IL-18, as well as the pore-forming protein gasdermin D (GSDMD). GSDMD is responsible for the release of the two cytokines and the induction of lytic and inflammatory cell death known as pyroptosis. Each inflammasome receptor detects specific stimuli, either directly or indirectly, thereby enhancing the cell's ability to sense infections or homeostatic disturbances. In this chapter, we present the activation mechanism of the so-called "canonical" inflammasomes.
Inflammation is a key characteristic of all stages of tumor development, including tumor initiation, progression, malignant transformation, invasion, and metastasis. Inflammasomes are an important component of the inflammatory response and an indispensable part of the innate immune system. Inflammasomes regulate the nature of infiltrating immune cells by signaling the secretion of different cytokines and chemokines, thus regulating the anti-tumor immunity of the body. Inflammasome expression patterns vary across different tumor types and stages, playing different roles during tumor progression. The complex diversity of the inflammasomes is determined by both internal and external factors relating to tumor establishment and progression. Therefore, elucidating the specific effects of different inflammasomes in anti-tumor immunity is critical for promoting the discovery of inflammasome-targeting drugs. This review focuses on the structure, activation pathway, and identification methods of the NLRP3, NLRC4, NLRP1 and AIM2 inflammasomes. Herein, we also explore the role of inflammasomes in different cancers and their complex regulatory mechanisms, and discuss current and future directions for targeting inflammasomes in cancer therapy. A detailed knowledge of inflammasome function and regulation may lead to novel therapies that target the activation of inflammasomes as well as the discovery of new drug targets.
Gram-positive bacterial infection causes high morbidity and mortality worldwide, while the underlying mechanism for host sensing bacterial components and initiating immune responses remains elusive. The NLRP3 inflammasome is a cytosolic multi-protein complex sensing a broad spectrum of endogenous danger signals and environmental irritants. In contrast to canonical NLRP3 inflammasome activation that needs both priming and activation signals, Lipopolysaccharide (LPS) from gram-negative bacteria activates the "one-step" NLRP3 inflammasome in human monocytes, which relies on the TLR4-TRIF-Caspase-8 signaling. Here, we show that in human monocytes, TLR2 agonists such as heat-killed gram-positive bacteria, peptidoglycan (PGN) or synthetic bacterial lipoprotein analog Pam3CysSerLys4 (Pam3CSK4) are able to induce the "one-step" NLRP3 inflammasome activation. Using genetic targeting and pharmacological inhibition approaches, it was found that TLR2 propagates signal through TRAF6, TAK1 and IKKβ, ultimately activated NLRP3 independent of RelA. In addition, IKKβ interacts with NLRP3 directly and affects NLRP3 inflammasome activation. These results reveal the signaling cascade downstream of TLR2 upon sensing gram-positive bacterial infection and activating the "one-step" NLRP3 inflammasome in human monocytes, which provides clue for controlling gram-positive bacterial infection-related inflammation.
Invasive fungal infections are associated with high mortality rates, and the lack of efficient treatment options emphasizes an urgency to identify underlying disease mechanisms. We report that disseminated Candida albicans infection is facilitated by interleukin-1 receptor antagonist (IL-1Ra) secreted from macrophages in two temporally and spatially distinct waves. Splenic CD169+ macrophages release IL-1Ra into the bloodstream, impeding early neutrophil recruitment. IL-1Ra secreted by monocyte-derived tissue macrophages further impairs pathogen containment. Therapeutic IL-1Ra neutralization restored the functional competence of neutrophils, corrected maladapted hyper-inflammation, and eradicated the otherwise lethal infection. Conversely, augmentation of macrophage-secreted IL-1Ra by type I interferon severely aggravated disease mortality. Our study uncovers how a fundamental immunoregulatory mechanism mediates the high disease susceptibility to invasive candidiasis. Furthermore, interferon-stimulated IL-1Ra secretion may exacerbate fungal dissemination in human patients with secondary candidemia. Macrophage-secreted IL-1Ra should be considered as an additional biomarker and potential therapeutic target in severe systemic candidiasis.
Atherosclerosis complicates chronic inflammatory diseases, such as rheumatoid arthritis and systemic lupus erythematosus, suggesting that a shared physiological pathway regulates inflammatory responses in these diseases wherein spleen tyrosine kinase (SYK) is involved. We aimed to identify a shared therapeutic target for atherosclerosis and inflammatory diseases. We used Syk-knockout atherosclerosis-prone mice to determine whether SYK is involved in atherosclerosis via the inflammatory response and elucidate the mechanism of SYK signaling. The Syk-knockout mice showed reduced atherosclerosis in vivo, and macrophages derived from this strain showed ameliorated cell migration in vitro. CD11c expression decreased on Syk-knockout monocytes and macrophages; it was upregulated by forkhead box protein O1 (FOXO1) after stimulation with granulocyte-macrophage colony-stimulating factor (GM-CSF), and c-Jun amino-terminal kinase (JNK) mediated SYK signaling to FOXO1. Furthermore, FOXO1 inhibitor treatment mitigated atherosclerosis in mice. Thus, GM-CSF receptor/SYK/JNK/FOXO1/CD11c signaling in monocytes and macrophages and FOXO1 could be therapeutic targets for atherosclerosis and inflammatory diseases.
Mycoplasma (M) hyopneumoniae causes enzootic pneumonia, a highly contagious respiratory disease in swine that causes significant economic losses worldwide. It is unknown whether the NLR family pyrin domain containing 3 (NLRP3) inflammasome regulates the immune response in swine during M. hyopneumoniae infection. The current study utilized an in vivo swine model of M. hyopneumoniae infection to investigate the regulatory functional role of the NLRP3 inflammasome during M. hyopneumoniae infection. Notable histopathological alterations were observed in M. hyopneumoniae-infected swine tissues, which were associated with an inflammatory response and disease progression. Swine M. hyopneumoniae infection was associated with an increase in the expression of the NLRP3 inflammasome, which stimulated pro-inflammatory cytokines such as tumour necrosis factor-alpha (TNF- α), interleukin 18 (IL-18), and interleukin 1 beta (IL-1 β). The impact of the NLRP3 inhibitor, MCC950 on NLRP3 and pro-inflammatory cytokines in M. hyopneumoniae-infected swine was examined to investigate the relationship between the NLRP3 inflammasome and M. hyopneumoniae infection. Taken together, our findings provide strong evidence that the NLRP3 inflammasome plays a critical regulatory functional role in M. hyopneumoniae infection in swine.
Candida albicans is a causative agent in mycoses of the skin, oral cavity, and gastrointestinal tract. Identification of receptors, and their respective ligands, that are engaged by immune cells when in contact with C. albicans is crucial for understanding inflammatory responses leading to invasive candidiasis. Mincle is a recently identified macrophage-expressed receptor that is important for host responses to C. albicans. The carbohydrate-recognition domain of human and mouse Mincle were expressed, purified under denaturing conditions, and successfully refolded. In addition to oligomers, there are isolatable monomeric and dimeric forms of the protein that occur under two different buffer solutions. The human and mouse homologues bound yeast extract, and the isolated dimeric and monomeric species also demonstrated the recognition of whole C. albicans yeast cells. The data are indicative of several functional states mediating the interaction of Mincle and yeast at the surface of the macrophage.
Caspase 1, formerly designated interleukin 1beta (IL-1beta)-converting enzyme, processes pro-IL-1beta and pro-IL-18 to yield active cytokines that play a pivotal role in inflammation and cell activation. We show here the effect of caspase 1 deficiency on the inflammatory and adaptive immune responses to the fungus Candida albicans. Caspase 1 deficiency did not affect susceptibility to primary systemic infection with the fungus, as revealed by survival and fungal growth. However, Th1-mediated resistance to reinfection was greatly impaired in caspase 1-deficient mice, and this correlated with low-level production of IL-12 and gamma interferon. Early in infection, production of these cytokines and that of tumor necrosis factor alpha, IL-6, and, interestingly, IL-1beta occurred normally in caspase 1-deficient mice, while that of IL-18 was severely impaired. Exogenous administration of IL-18, more than IL-12, restored the Th1-mediated resistance to the infection. We conclude that, while caspase 1 is not indispensable for release of mature IL-1beta in candidiasis, the caspase 1-dependent production of IL-18 may represent an important and novel pathway for the expression of sustained Th1 reactivity to the fungus.
A crucial part of the innate immune response is the assembly of the inflammasome, a cytosolic complex of proteins that activates caspase-1 to process the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The adaptor protein ASC is essential for inflammasome function, binding directly to caspase-1 (refs 3, 4), but the triggers of this interaction are less clear. ASC also interacts with the adaptor cryopyrin (also known as NALP3 or CIAS1). Activating mutations in cryopyrin are associated with familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal onset multisystem inflammatory disease, diseases that are characterized by excessive production of IL-1beta. Here we show that cryopyrin-deficient macrophages cannot activate caspase-1 in response to Toll-like receptor agonists plus ATP, the latter activating the P2X7 receptor to decrease intracellular K+ levels. The release of IL-1beta in response to nigericin, a potassium ionophore, and maitotoxin, a potent marine toxin, was also found to be dependent on cryopyrin. In contrast to Asc-/- macrophages, cells deficient in the gene encoding cryopyrin (Cias1-/-) activated caspase-1 and secreted normal levels of IL-1beta and IL-18 when infected with Gram-negative Salmonella typhimurium or Francisella tularensis. Macrophages exposed to Gram-positive Staphylococcus aureus or Listeria monocytogenes, however, required both ASC and cryopyrin to activate caspase-1 and secrete IL-1beta. Therefore, cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes.
Development of the acute and chronic inflammatory responses known as gout and pseudogout are associated with the deposition of monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals, respectively, in joints and periarticular tissues. Although MSU crystals were first identified as the aetiological agent of gout in the eighteenth century and more recently as a 'danger signal' released from dying cells, little is known about the molecular mechanisms underlying MSU- or CPPD-induced inflammation. Here we show that MSU and CPPD engage the caspase-1-activating NALP3 (also called cryopyrin) inflammasome, resulting in the production of active interleukin (IL)-1beta and IL-18. Macrophages from mice deficient in various components of the inflammasome such as caspase-1, ASC and NALP3 are defective in crystal-induced IL-1beta activation. Moreover, an impaired neutrophil influx is found in an in vivo model of crystal-induced peritonitis in inflammasome-deficient mice or mice deficient in the IL-1beta receptor (IL-1R). These findings provide insight into the molecular processes underlying the inflammatory conditions of gout and pseudogout, and further support a pivotal role of the inflammasome in several autoinflammatory diseases.
The pathogenesis of Bacillus anthracis, the bacterium that causes anthrax, depends on secretion of three factors that combine to form two bipartite toxins. Edema toxin, consisting of protective antigen (PA) and edema factor (EF), causes the edema associated with cutaneous anthrax infections, whereas lethal toxin (LeTx), consisting of PA and lethal factor (LF), is believed to be responsible for causing death in systemic anthrax infections. EF and LF can be transported by PA into the cytosol of many cell types. In mouse macrophages, LF can cause rapid necrosis that may be related to the pathology of systemic infections. Inbred mouse strains display variable sensitivity to LeTx-induced macrophage necrosis. This trait difference has been mapped to a locus on chromosome 11 named Ltxs1 (refs. 7,8). Here we show that an extremely polymorphic gene in this locus, Nalp1b, is the primary mediator of mouse macrophage susceptibility to LeTx. We also show that LeTx-induced macrophage death requires caspase-1, which is activated in susceptible, but not resistant, macrophages after intoxication, suggesting that Nalp1b directly or indirectly activates caspase-1 in response to LeTx.
Interleukin (IL)-1 alpha and IL-1 beta are protective proinflammatory cytokines involved in host defense against Candida albicans. It is, however, unknown whether they provide protection through similar mechanisms. We investigated the effect of endogenous IL-1 alpha and IL-1 beta on disseminated C. albicans infection.
Mice deficient in the genes encoding IL-1 alpha (IL-1 alpha-/-), IL-1 beta (IL-1 beta-/-), or both molecules (IL-1 alpha-/- beta-/-) were used. Survival and C. albicans outgrowth in the kidneys was assessed after intravenous injection of C. albicans.
Both mortality and C. albicans outgrowth in the kidneys were significantly increased in IL-1 alpha-/- and IL-1 beta-/- mice, compared with those in control mice, with the IL-1 alpha-/- beta-/- mice being most susceptible to disseminated candidiasis. The host defense mechanisms triggered by IL-1 alpha and IL-1 beta differed from one another. IL-1 beta-/- mice showed decreased recruitment of granulocytes in response to an intraperitoneal C. albicans challenge, and generation of superoxide production was diminished in IL-1 beta-/- granulocytes. IL-1 alpha-/- mice had a reduced capacity to damage C. albicans pseudohyphae. Protective type 1 responses were deficient in both IL-1 alpha-/- and IL-1 beta-/- mice, as assessed by production of interferon-gamma by splenocytes in response to heat-killed C. albicans.
Although IL-1 alpha and IL-1 beta have differential effects on the various arms of host defense, both cytokines are essential for mounting a protective host response against invasive C. albicans infection.
Fungal infections are increasing worldwide due to the marked rise in immunodeficiencies including AIDS; however, immune responses to fungi are poorly understood. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 represents the prototype of innate non-Toll-like receptors (TLRs) containing immunoreceptor tyrosine-based activation motifs (ITAMs) related to those of adaptive antigen receptors. Here we identify Card9 as a key transducer of Dectin-1 signalling. Although being dispensable for TLR/MyD88-induced responses, Card9 controls Dectin-1-mediated myeloid cell activation, cytokine production and innate anti-fungal immunity. Card9 couples to Bcl10 and regulates Bcl10-Malt1-mediated NF-kappaB activation induced by zymosan. Yet, Card9 is dispensable for antigen receptor signalling that uses Carma1 as a link to Bcl10-Malt1. Thus, our results define a novel innate immune pathway and indicate that evolutionarily distinct ITAM receptors in innate and adaptive immune cells use diverse adaptor proteins to engage selectively the conserved Bcl10-Malt1 module.
Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.
C-type lectins expressed on myeloid cells comprise a family of proteins that share a common structural motif, and some act as receptors in pathogen recognition. But just as the presence of leucine-rich repeats alone is not sufficient to define a Toll-like receptor, the characterization of C-type lectin receptors in innate immunity requires the identification of accompanying signaling motifs. Here we focus on the known signaling pathways of myeloid C-type lectins and on their possible functions as autonomous activating or inhibitory receptors involved in innate responses to pathogens or self.
Secretion of the proinflammatory cytokines, interleukin (IL)-1beta and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1beta and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines.
The C-type lectin dectin-1 binds to yeast and signals through the kinase Syk and the adaptor CARD9 to induce production of interleukin 10 (IL-10) and IL-2 in dendritic cells (DCs). However, whether this pathway promotes full DC activation remains unclear. Here we show that dectin-1-Syk-CARD9 signaling induced DC maturation and the secretion of proinflammatory cytokines, including IL-6, tumor necrosis factor and IL-23, but little IL-12. Dectin-1-activated DCs 'instructed' the differentiation of CD4+ IL-17-producing effector T cells (T(H)-17 cells) in vitro, and a dectin-1 agonist acted as an adjuvant promoting the differentiation of T(H)-17 and T helper type 1 cells in vivo. Infection with Candida albicans induced CARD9-dependent T(H)-17 responses to the organism. Our data indicate that signaling through Syk and CARD9 can couple innate to adaptive immunity independently of Toll-like receptor signals and that CARD9 is required for the development of T(H)-17 responses to some pathogens.
Immunoreceptor tyrosine-based activation motifs (ITAMs) are crucial in antigen receptor signaling in acquired immunity. Although receptors associated with the ITAM-bearing adaptors FcRgamma and DAP12 on myeloid cells have been suggested to activate innate immune responses, the mechanism coupling those receptors to 'downstream' signaling events is unclear. The CARMA1-Bcl-10-MALT1 complex is critical for the activation of transcription factor NF-kappaB in lymphocytes but has an unclear function in myeloid cells. Here we report that deletion of the gene encoding the Bcl-10 adaptor-binding partner CARD9 resulted in impaired myeloid cell activation of NF-kappaB signaling by several ITAM-associated receptors. Moreover, CARD9 was required for Toll-like receptor-induced activation of dendritic cells through the activation of mitogen-activated protein kinases. Although Bcl10-/- and Card9-/- mice had similar signaling impairment in myeloid cells, Card11-/- (CARMA1-deficient) myeloid cell responses were normal, and although Card11-/- lymphocytes were defective in antigen receptor-mediated activation, Card9-/- lymphocytes were not. Thus, the activation of lymphoid and myeloid cells through ITAM-associated receptors or Toll-like receptors is regulated by CARMA1-Bcl-10 and CARD9-Bcl-10, respectively.
Inflammasomes are Nod-like receptor(NLR)- and caspase-1-containing cytoplasmic multiprotein complexes, which upon their assembly, process and activate the proinflammatory cytokines interleukin (IL)-1beta and IL-18. The inflammasomes harboring the NLR members NALP1, NALP3 and IPAF have been best characterized. While the IPAF inflammasome is activated by bacterial flagellin, activation of the NALP3 inflammasome is triggered not only by several microbial components, but also by a plethora of danger-associated host molecules such as uric acid. How NALP3 senses these chemically unrelated activators is not known. Here, we provide evidence that activation of NALP3, but not of the IPAF inflammasome, is blocked by inhibiting K(+) efflux from cells. Low intracellular K(+) is also a requirement for NALP1 inflammasome activation by lethal toxin of Bacillus anthracis. In vitro, NALP inflammasome assembly and caspase-1 recruitment occurs spontaneously at K(+) concentrations below 90 mM, but is prevented at higher concentrations. Thus, low intracellular K(+) may be the least common trigger of NALP-inflammasome activation.
The innate immune system recognizes nucleic acids during infection and tissue damage. Whereas viral RNA is detected by endosomal toll-like receptors (TLR3, TLR7, TLR8) and cytoplasmic RIG-I and MDA5, endosomal TLR9 and cytoplasmic DAI bind DNA, resulting in the activation of nuclear factor-kappaB and interferon regulatory factor transcription factors. However, viruses also trigger pro-inflammatory responses, which remain poorly defined. Here we show that internalized adenoviral DNA induces maturation of pro-interleukin-1beta in macrophages, which is dependent on NALP3 and ASC, components of the innate cytosolic molecular complex termed the inflammasome. Correspondingly, NALP3- and ASC-deficient mice display reduced innate inflammatory responses to adenovirus particles. Inflammasome activation also occurs as a result of transfected cytosolic bacterial, viral and mammalian (host) DNA, but in this case sensing is dependent on ASC but not NALP3. The DNA-sensing pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors. Thus, in addition to viral and bacterial components or danger signals in general, inflammasomes sense potentially dangerous cytoplasmic DNA, strengthening their central role in innate immunity.
Expression levels of the papain-like cysteine protease cathepsin B (Ctsb) have been positively correlated with mammary tumour progression and metastasis; however, its roles in the hallmark processes of malignant growth remain poorly defined. Using Ctsb-deficient mice we investigated tumour cell differentiation, proliferation and apoptosis in the Tg(MMTV-PyMT) mouse mammary cancer model. Absence of Ctsb significantly impaired development of high-grade invasive ductal carcinomas and reduced the metastatic burden in the lungs. Mice lacking Ctsb exhibited reduced cell proliferation in mammary carcinomas and their lung metastases. Notably, intravenous injection of primarily isolated, Ctsb-expressing tumour cells into congenic Ctsb-deficient mice revealed impaired cell proliferation in the resulting experimental lung metastases, providing evidence for the involvement of Ctsb in paracrine regulation of cancer cell proliferation. No Ctsb genotype-dependent difference in tumour cell death was observed in vivo or by treatment of isolated PyMT cancer cells with tumour necrosis factor-alpha. However, cancer cells lacking Ctsb exhibited significantly higher resistance to apoptosis induction by the lysosomotropic agent Leu-Leu-OMe. Thus, our results indicate an in vivo role for Ctsb in promoting cellular anaplasia in mammary cancers and proliferation in lung metastases.
The inhalation of airborne pollutants, such as asbestos or silica, is linked to inflammation of the lung, fibrosis, and lung
cancer. How the presence of pathogenic dust is recognized and how chronic inflammatory diseases are triggered are poorly understood.
Here, we show that asbestos and silica are sensed by the Nalp3 inflammasome, whose subsequent activation leads to interleukin-1β
secretion. Inflammasome activation is triggered by reactive oxygen species, which are generated by a NADPH oxidase upon particle
phagocytosis. (NADPH is the reduced form of nicotinamide adenine dinucleotide phosphate.) In a model of asbestos inhalation,
Nalp3–/– mice showed diminished recruitment of inflammatory cells to the lungs, paralleled by lower cytokine production. Our findings
implicate the Nalp3 inflammasome in particulate matter–related pulmonary diseases and support its role as a major proinflammatory
“danger” receptor.
Dengue haemorrhagic fever and dengue shock syndrome, the most severe responses to dengue virus (DV) infection, are characterized by plasma leakage (due to increased vascular permeability) and low platelet counts. CLEC5A (C-type lectin domain family 5, member A; also known as myeloid DAP12-associating lectin (MDL-1)) contains a C-type lectin-like fold similar to the natural-killer T-cell C-type lectin domains and associates with a 12-kDa DNAX-activating protein (DAP12) on myeloid cells. Here we show that CLEC5A interacts with the dengue virion directly and thereby brings about DAP12 phosphorylation. The CLEC5A-DV interaction does not result in viral entry but stimulates the release of proinflammatory cytokines. Blockade of CLEC5A-DV interaction suppresses the secretion of proinflammatory cytokines without affecting the release of interferon-alpha, supporting the notion that CLEC5A acts as a signalling receptor for proinflammatory cytokine release. Moreover, anti-CLEC5A monoclonal antibodies inhibit DV-induced plasma leakage, as well as subcutaneous and vital-organ haemorrhaging, and reduce the mortality of DV infection by about 50% in STAT1-deficient mice. Our observation that blockade of CLEC5A-mediated signalling attenuates the production of proinflammatory cytokines by macrophages infected with DV (either alone or complexed with an enhancing antibody) offers a promising strategy for alleviating tissue damage and increasing the survival of patients suffering from dengue haemorrhagic fever and dengue shock syndrome, and possibly even other virus-induced inflammatory diseases.
Inhalation of silica crystals causes inflammation in the alveolar space. Prolonged exposure to silica can lead to the development of silicosis, an irreversible, fibrotic pulmonary disease. The mechanisms by which silica and other crystals activate immune cells are not well understood. Here we demonstrate that silica and aluminum salt crystals activated inflammasomes formed by the cytoplasmic receptor NALP3. NALP3 activation required phagocytosis of crystals, and this uptake subsequently led to lysosomal damage and rupture. 'Sterile' lysosomal damage (without crystals) also induced NALP3 activation, and inhibition of either phagosomal acidification or cathepsin B activity impaired NALP3 activation. Our results indicate that the NALP3 inflammasome senses lysosomal damage as an endogenous 'danger' signal.
The fibrillar peptide amyloid-beta (A beta) has a chief function in the pathogenesis of Alzheimer's disease. Interleukin 1 beta (IL-1 beta) is a key cytokine in the inflammatory response to A beta. Insoluble materials such as crystals activate the inflammasome formed by the cytoplasmic receptor NALP3, which results in the release of IL-1 beta. Here we identify the NALP3 inflammasome as a sensor of A beta in a process involving the phagocytosis of A beta and subsequent lysosomal damage and release of cathepsin B. Furthermore, the IL-1 beta pathway was essential for the microglial synthesis of proinflammatory and neurotoxic factors, and the inflammasome, caspase-1 and IL-1 beta were critical for the recruitment of microglia to exogenous A beta in the brain. Our findings suggest that activation of the NALP3 inflammasome is important for inflammation and tissue damage in Alzheimer's disease.
The topic of immunity to fungal infections is of interest to a wide range of disciplines, from microbiology to immunology. It is of particular interest in terms of therapy of HIV-infected individuals, and patients with cancer or individuals who have received transplants. Understanding the nature and function of the immune response to fungi is an exciting challenge that might set the stage for new approaches to the treatment of fungal diseases, from immunotherapy to vaccines. The past decade has witnessed the development of a wide range of new approaches to elucidate events that occur at the host-fungus interface.
IL-1β-converting enzyme (ICE) cleaves pro-1L-1β to generate mature IL-1β. ICE is homologous to other proteins that have been implicated in apoptosis, including CED-3 and Nedd-2/Ich-1. We generated ICE-deficient mice and observed that they are overtly normal but have a major defect in the production of mature IL-1β after stimulation with lipopolysaccharide. IL-1α production is also impaired. ICE-deficient mice are resistant to endotoxic shock. Thymocytes and macrophages from the ICE-deficient animals undergo apoptosis normally. ICE therefore plays a dominant role in the generation of mature IL-1β, a previously unsuspected role in production of IL-1α, but has no autonomous function in apoptosis.
Fungal diseases represent an important paradigm in immunology, as they can result from either a lack of recognition by the immune system or overactivation of the inflammatory response. Research in this field is entering an exciting period of transition from studying the molecular and cellular bases of fungal virulence to determining the cellular and molecular mechanisms that maintain immune homeostasis with fungi. The fine line between these two research areas is central to our understanding of tissue homeostasis and its possible breakdown in fungal infections and diseases. Recent insights into immune responses to fungi suggest that functionally distinct mechanisms have evolved to achieve optimal host-fungus interactions in mammals.
Binding of particulate antigens by antigen-presenting cells is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. By using atomic force microscopy as a tool for real-time single-cell activation analysis, we report that uric acid crystals could directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein-based cellular contacts. Binding of particulate substances activated Syk kinase-dependent signaling in dendritic cells. These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell-surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation, and adjuvanticity.
Spleen tyrosine kinase (Syk) has been identified as an important modulator of immune signaling in B cells and cells bearing Fcgamma-activating receptors. R788, a prodrug of active metabolite R406, has been shown to be an inhibitor of Syk kinase, active in a variety of in vitro and in vivo models, suggesting potential activity in the treatment of rheumatoid arthritis (RA).
We enrolled 189 patients with active RA despite methotrexate therapy in a 3-month, multicenter, ascending-dose, double-blind, placebo-controlled trial. The primary end point was the American College of Rheumatology 20% improvement criteria (ACR20) response rate at week 12.
Twice-daily oral doses of 100 mg and 150 mg of R788 were significantly superior to placebo or twice-daily oral doses of 50 mg at week 12 (ACR20 achieved in 65% and 72% versus 38% and 32% of patients, respectively [P < 0.01]). ACR50 (achieved in 49% and 57% versus 19% and 17% of patients, respectively) and ACR70 (achieved in 33% and 40% versus 4% and 2% of patients, respectively) scores showed a similar pattern. Clinical effect was noted as early as 1 week after initiation of therapy. Reductions in serum interleukin-6 and matrix metalloproteinase 3 levels also occurred as early as week 1 in the groups receiving 100 mg and 150 mg R788. The major adverse effects were gastrointestinal side effects (predominantly diarrhea) and neutropenia (<1,500/mm3), both of which were dose related.
These results indicate that an inhibitor of Syk kinase produces significant clinical benefits at 12 weeks in a population of patients with active RA receiving methotrexate therapy. Syk kinase may be an important new therapeutic target in RA and related autoimmune conditions.
The inflammasome is a large multiprotein complex whose assembly leads to the activation of caspase-1, which promotes the maturation of proinflammatory cytokines interleukin-1beta (IL-1beta) and IL-18. Proteins encoded by the nucleotide-binding domain and leucine-rich repeat (NLR) containing gene family form the central components of inflammasomes and act as intracellular sensors to detect cytosolic microbial components and "danger" signals (such as ATP and toxins). The inflammasome not only plays a pivotal role in innate immune responses toward pathogens but also mediates the activity of aluminum adjuvants. Thus, the inflammasome and associated signaling pathways are attractive targets for new therapeutics and vaccines.
Macrophage-inducible C-type lectin (Mincle) is expressed mainly in macrophages and is induced after exposure to various stimuli and stresses. Here we show that Mincle selectively associated with the Fc receptor common gamma-chain and activated macrophages to produce inflammatory cytokines and chemokines. Mincle-expressing cells were activated in the presence of dead cells, and we identified SAP130, a component of small nuclear ribonucloprotein, as a Mincle ligand that is released from dead cells. To investigate whether Mincle is required for normal responses to cell death in vivo, we induced thymocyte death by irradiating mice and found that transient infiltration of neutrophils into the thymus could be blocked by injection of Mincle-specific antibody. Our results suggest that Mincle is a receptor that senses nonhomeostatic cell death and thereby induces the production of inflammatory cytokines to drive the infiltration of neutrophils into damaged tissue.
MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.
Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively. The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the 'inflammasome', a complex comprising several adaptors. Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function. ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages. Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1beta and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock. Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages. Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S. typhimurium. Caspase-1 activation was not compromised by loss of RIP2. These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens. Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.
Dectin-1 is a lectin receptor for beta-glucan that is important for innate macrophage recognition of fungi and contributes to phagocytosis, reactive oxygen production, and induction of inflammatory cytokines. The mechanisms by which Dectin-1 mediates intracellular signaling are just beginning to be defined. Spleen tyrosine kinase (Syk) is a protein tyrosine kinase that is critical for adaptive immune responses where it mediates signaling through B-cell receptors, T-cell receptors, and Fc receptors. Here we report that Dectin-1 activates Syk in macrophages and is important for Dectin-1-stimulated reactive oxygen production, but not for phagocytosis. Syk activation is restricted to a subpopulation of macrophages that is in equilibrium with cells that cannot activate the pathway. The proportion of macrophages using this signaling pathway can be modulated by cytokine treatment. Thus, Dectin-1 signaling reveals dynamic macrophage heterogeneity in inflammatory activation potential.
Spleen tyrosine kinase (Syk), a key mediator of immunoreceptor signaling in inflammatory cells, is essential for immune complex-mediated signal transduction initiated by activated receptors for immunoglobulin G. In collagen-induced arthritis, R788/R406, a novel and potent small molecule Syk inhibitor suppressed clinical arthritis, bone erosions, pannus formation, and synovitis. Serum anti-collagen type II antibody levels were unaltered, while the half-life of exogenous antibody was extended when co-administered with R406. Expression of the targeted kinase (Syk) in synovial tissue correlated with the joint level of inflammatory cell infiltrates and was virtually undetectable in treated rats. Syk inhibition suppressed synovial cytokines and cartilage oligomeric matrix protein (COMP) in serum, suggesting a sensitive and reliable biomarker for R406 activity. These results highlight the role of activating Fcgamma receptors in inflammatory synovitis and suggest that interruption of the signaling cascade with a novel Syk inhibitor may be a useful addition to immunosuppressive disease-modifying anti-rheumatic drugs currently used in the treatment of human autoimmune diseases such as rheumatoid arthritis.
Myeloid C-type lectins in innate immunity
- M J Robinson
- MJ Robinson