The epidemiology of invasive fungal infections in transplant recipients.

Transplant patients, including solid-organ transplant (SOT) and hematopoietic stem cell transplant (HSCT) recipients, are exposed to various types of complications, particularly rejection. To prevent these outcomes, transplant recipients commonly receive long-term immunosuppressive regimens that in turn make them more susceptible to a wide array of infectious diseases, notably those caused by opportunistic pathogens. Among these, invasive fungal infections (IFIs) remain a major cause of mortality and morbidity in both SOT and HSCT recipients. Despite the continuing improvement in early diagnostics and treatments of IFIs, the management of these infections in transplant patients is still complicated. Here, we provide an overview concerning the most recent trends in the epidemiology of IFIs in SOT and HSCT recipients by describing the prominent yeast and mold species involved, the timing of post-transplant IFIs and the risk factors associated with their occurrence in these particularly weak populations. We also give special emphasis into basic research advances in the field that recently suggested a role of the global and long-term prophylactic regimen in orchestrating various biological disturbances in the organism and conditioning the emergence of the most adapted fungal strains to the particular physiological profiles of transplant patients.

Extracellular vesicles: new bullets in the fungal armory.

Invasive fungal infections represent millions of deaths per year, but their pathophysiology remains insufficiently understood. Host-fungi interplay has been recently shown to include extracellular vesicles derived from fungi and host infected cells. In this forum article we discuss their emerging role in modulating the host immune response with particular emphasis on their regulatory involvement during Candida albicans infection.

Preventive nasal administration of flagellin restores antimicrobial effect of gentamicin and protects against a multidrug-resistant strain of Pseudomonas aeruginosa.

The increasing prevalence of multidrug-resistant Pseudomonas aeruginosa (PA) is a significant concern for chronic respiratory disease exacerbations. Host-directed drugs, such as flagellin, an agonist of toll-like receptor 5 (TLR5), have emerged as a promising solution. In this study, we evaluated the prophylactic intranasal administration of flagellin against a multidrug-resistant strain of PA (PAMDR) in mice and assessed the possible synergy with the antibiotic gentamicin (GNT). The results indicated that flagellin treatment before infection decreased bacterial load in the lungs, likely due to an increase in neutrophil recruitment, and reduced signs of inflammation, including proinflammatory cytokines. The combination of flagellin and GNT showed a synergistic effect, decreasing even more the bacterial load and increasing mice survival rates, in comparison to mice pre-treated only with flagellin. These findings suggest that preventive nasal administration of flagellin could restore the effect of GNT against MDR strains of PA, paving the way for the use of flagellin in vulnerable patients with chronic respiratory diseases.

Specific NLRP3 Inflammasome Assembling and Regulation in Neutrophils: Relevance in Inflammatory and Infectious Diseases.

The NLRP3 inflammasome is a cytosolic multimeric protein platform that leads to the activation of the protease zymogen, caspase-1 (CASP1). Inflammasome activation mediates the proteolytic activation of pro-inflammatory cytokines (IL-1ß and IL-18) and program cell death called pyroptosis. The pyroptosis is mediated by the protein executioner Gasdermin D (GSDMD), which forms pores at the plasma membrane to facilitate IL-1ß/IL-18 secretion and causes pyroptosis. The NLRP3 inflammasome is activated in response to a large number of pathogenic and sterile insults. However, an uncontrolled inflammasome activation may drive inflammation-associated diseases. Initially, inflammasome-competent cells were believed to be limited to macrophages, dendritic cells (DC), and monocytes. However, emerging evidence indicates that neutrophils can assemble inflammasomes in response to various stimuli with functional relevance. Interestingly, the regulation of inflammasome in neutrophils appears to be unconventional. This review provides a broad overview of the role and regulation of inflammasomes-and more specifically NLRP3-in neutrophils.

The IFN-inducible GTPase IRGB10 regulates viral replication and inflammasome activation during influenza A virus infection in mice.

The upregulation of interferon (IFN)-inducible GTPases in response to pathogenic insults is vital to host defense against many bacterial, fungal, and viral pathogens. Several IFN-inducible GTPases play key roles in mediating inflammasome activation and providing host protection after bacterial or fungal infections, though their role in inflammasome activation after viral infection is less clear. Among the IFN-inducible GTPases, the expression of immunity-related GTPases (IRGs) varies widely across species for unknown reasons. Here, we report that IRGB10, but not IRGM1, IRGM2, or IRGM3, is required for NLRP3 inflammasome activation in response to influenza A virus (IAV) infection in mice. While IRGB10 functions to release inflammasome ligands in the context of bacterial and fungal infections, we found that IRGB10 facilitates endosomal maturation and nuclear translocation of IAV, thereby regulating viral replication. Corresponding with our in vitro results, we found that Irgb10-/- mice were more resistant to IAV-induced mortality than WT mice. The results of our study demonstrate a detrimental role of IRGB10 in host immunity in response to IAV and a novel function of IRGB10, but not IRGMs, in promoting viral translocation into the nucleus.

Fungal cell wall components modulate our immune system.

Invasive fungal infections remain highly problematic for human health. Collectively, they account for more than 1 million deaths a year in addition to more than 100 million mucosal infections and 1 billion skin infections. To be able to make progress it is important to understand the pathobiology of fungal interactions with the immune system. Here, we highlight new advancements pointing out the pivotal role of fungal cell wall components (ß-glucan, mannan, galactosaminogalactan and melanin) in modulating host immunity and discuss how these open new opportunities for the development of immunomodulatory strategies to combat deadly fungal infectious diseases.

DDX3X coordinates host defense against influenza virus by activating the NLRP3 inflammasome and type I interferon response.

Viruses and hosts have coevolved for millions of years, leading to the development of complex host-pathogen interactions. Influenza A virus (IAV) causes severe pulmonary pathology and is a recurrent threat to human health. Innate immune sensing of IAV triggers a complex chain of host responses. IAV has adapted to evade host defense mechanisms, and the host has coevolved to counteract these evasion strategies. However, the molecular mechanisms governing the balance between host defense and viral immune evasion is poorly understood. Here, we show that the host protein DEAD-box helicase 3 X-linked (DDX3X) is critical to orchestrate a multifaceted antiviral innate response during IAV infection, coordinating the activation of the nucleotide-binding oligomerization domain-like receptor with a pyrin domain 3 (NLRP3) inflammasome, assembly of stress granules, and type I interferon (IFN) responses. DDX3X activated the NLRP3 inflammasome in response to WT IAV, which carries the immune evasive nonstructural protein 1 (NS1). However, in the absence of NS1, DDX3X promoted the formation of stress granules that facilitated efficient activation of type I IFN signaling. Moreover, induction of DDX3X-containing stress granules by external stimuli after IAV infection led to increased type I IFN signaling, suggesting that NS1 actively inhibits stress granule-mediated host responses and DDX3X-mediated NLRP3 activation counteracts this action. Furthermore, the loss of DDX3X expression in myeloid cells caused severe pulmonary pathogenesis and morbidity in IAV-infected mice. Together, our findings show that DDX3X orchestrates alternate modes of innate host defense which are critical to fight against NS1-mediated immune evasion strategies during IAV infection.

Galactosaminogalactan activates the inflammasome to provide host protection.

Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.

ZBP1 promotes fungi-induced inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis).

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens.

Identification of the PANoptosome: A Molecular Platform Triggering Pyroptosis, Apoptosis, and Necroptosis (PANoptosis).

Programmed cell death plays crucial roles in organismal development and host defense. Recent studies have highlighted mechanistic overlaps and extensive, multifaceted crosstalk between pyroptosis, apoptosis, and necroptosis, three programmed cell death pathways traditionally considered autonomous. The growing body of evidence, in conjunction with the identification of molecules controlling the concomitant activation of all three pathways by pathological triggers, has led to the development of the concept of PANoptosis. During PANoptosis, inflammatory cell death occurs through the collective activation of pyroptosis, apoptosis, and necroptosis, which can circumvent pathogen-mediated inhibition of individual death pathways. Many of the molecular details of this emerging pathway are unclear. Here, we describe the activation of PANoptosis by bacterial and viral triggers and report protein interactions that reveal the formation of a PANoptosome complex. Infection of macrophages with influenza A virus, vesicular stomatitis virus, Listeria monocytogenes, or Salmonella enterica serovar Typhimurium resulted in robust cell death and the hallmarks of PANoptosis activation. Combined deletion of the PANoptotic components caspase-1 (CASP1), CASP11, receptor-interacting serine/threonine-protein kinase 3 (RIPK3), and CASP8 largely protected macrophages from cell death induced by these pathogens, while deletion of individual components provided reduced or no protection. Further, molecules from the pyroptotic, apoptotic, and necroptotic cell death pathways interacted to form a single molecular complex that we have termed the PANoptosome. Overall, our study identifies pathogens capable of activating PANoptosis and the formation of a PANoptosome complex.

Interferon inducible GBPs restrict Burkholderia thailandensis motility induced cell-cell fusion.

Innate immunity responds to pathogens by producing alarm signals and activating pathways that make host cells inhospitable for pathogen replication. The intracellular bacterium Burkholderia thailandensis invades the cytosol, hijacks host actin, and induces cell fusion to spread to adjacent cells, forming multinucleated giant cells (MNGCs) which promote bacterial replication. We show that type I interferon (IFN) restricts macrophage MNGC formation during B. thailandensis infection. Guanylate-binding proteins (GBPs) expressed downstream of type I IFN were required to restrict MNGC formation through inhibition of bacterial Arp2/3-dependent actin motility during infection. GTPase activity and the CAAX prenylation domain were required for GBP2 recruitment to B. thailandensis, which restricted bacterial actin polymerization required for MNGC formation. Consistent with the effects in in vitro macrophages, Gbp2-/-, Gbp5-/-, GbpChr3-KO mice were more susceptible to intranasal infection with B. thailandensis than wildtype mice. Our findings reveal that IFN and GBPs play a critical role in restricting cell-cell fusion and bacteria-induced pathology during infection.

DNA Sensing in the Innate Immune Response.

The innate immune system recognizes conserved pathogen-associated molecular patterns and produces inflammatory cytokines that direct downstream immune responses. The inappropriate localization of DNA within the cell cytosol or endosomal compartments indicates that a cell may either be infected by a DNA virus or bacterium, or has problems with its own nuclear integrity. This DNA is sensed by certain receptors that mediate cytokine production and, in some cases, initiate an inflammatory and lytic form of cell death called pyroptosis. Dysregulation of these DNA-sensing pathways is thought to contribute to autoimmune diseases and the development of cancer. In this review, we will discuss the DNA sensors Toll-like receptor 9 (TLR9), cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), absent in melanoma 2 (AIM2), and interferon gamma-inducible 16 (IFI16), their ligands, and their physiological significance. We will also examine the less-well-understood DEAH- and DEAD-box helicases DHX9, DHX36, DDX41, and RNA polymerase III, each of which may play an important role in DNA-mediated innate immunity.

Hemagglutinin Stability Regulates H1N1 Influenza Virus Replication and Pathogenicity in Mice by Modulating Type I Interferon Responses in Dendritic Cells.

Hemagglutinin (HA) stability, or the pH at which HA is activated to cause membrane fusion, has been associated with the replication, pathogenicity, transmissibility, and interspecies adaptation of influenza A viruses. Here, we investigated the mechanisms by which a destabilizing HA mutation, Y17H (activation pH, 6.0), attenuates virus replication and pathogenicity in DBA/2 mice compared to wild-type (WT) virus (activation pH, 5.5). The extracellular lung pH was measured to be near neutral (pH 6.9 to 7.5). WT and Y17H viruses had similar environmental stability at pH 7.0; thus, extracellular inactivation was unlikely to attenuate the Y17H virus. The Y17H virus had accelerated replication kinetics in MDCK, A549, and RAW 264.7 cells when inoculated at a multiplicity of infection (MOI) of 3 PFU/cell. The destabilizing mutation also increased early infectivity and type I interferon (IFN) responses in mouse bone marrow-derived dendritic cells (DCs). In contrast, the HA-Y17H mutation reduced virus replication in murine airway murine nasal epithelial cell and murine tracheal epithelial cell cultures and attenuated virus replication, virus spread, the severity of infection, and cellular infiltration in the lungs of mice. Normalizing virus infection and weight loss in mice by inoculating them with Y17H virus at a dose 500-fold higher than that of WT virus revealed that the destabilized mutant virus triggered the upregulation of more host genes and increased type I IFN responses and cytokine expression in DBA/2 mouse lungs. Overall, HA destabilization decreased virulence in mice by boosting early infection in DCs, resulting in the greater activation of antiviral responses, including the type I IFN response. These studies reveal that HA stability may regulate pathogenicity by modulating IFN responses.IMPORTANCE Diverse influenza A viruses circulate in wild aquatic birds, occasionally infecting farm animals. Rarely, an avian- or swine-origin influenza virus adapts to humans and starts a pandemic. Seasonal and many universal influenza vaccines target the HA surface protein, which is a key component of pandemic influenza viruses. Understanding the HA properties needed for replication and pathogenicity in mammals may guide response efforts to control influenza. Some antiviral drugs and broadly reactive influenza vaccines that target the HA protein have suffered resistance due to destabilizing HA mutations that do not compromise replicative fitness in cell culture. Here, we show that despite not compromising fitness in standard cell cultures, a destabilizing H1N1 HA stalk mutation greatly diminishes viral replication and pathogenicity in vivo by modulating type I IFN responses. This encourages targeting the HA stalk with antiviral drugs and vaccines as well as reevaluating previous candidates that were susceptible to destabilizing resistance mutations.

DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome.

The cellular stress response has a vital role in regulating homeostasis by modulating cell survival and death. Stress granules are cytoplasmic compartments that enable cells to survive various stressors. Defects in the assembly and disassembly of stress granules are linked to neurodegenerative diseases, aberrant antiviral responses and cancer1-5. Inflammasomes are multi-protein heteromeric complexes that sense molecular patterns that are associated with damage or intracellular pathogens, and assemble into cytosolic compartments known as ASC specks to facilitate the activation of caspase-1. Activation of inflammasomes induces the secretion of interleukin (IL)-1ß and IL-18 and drives cell fate towards pyroptosis-a form of programmed inflammatory cell death that has major roles in health and disease6-12. Although both stress granules and inflammasomes can be triggered by the sensing of cellular stress, they drive contrasting cell-fate decisions. The crosstalk between stress granules and inflammasomes and how this informs cell fate has not been well-studied. Here we show that the induction of stress granules specifically inhibits NLRP3 inflammasome activation, ASC speck formation and pyroptosis. The stress granule protein DDX3X interacts with NLRP3 to drive inflammasome activation. Assembly of stress granules leads to the sequestration of DDX3X, and thereby the inhibition of NLRP3 inflammasome activation. Stress granules and the NLRP3 inflammasome compete for DDX3X molecules to coordinate the activation of innate responses and subsequent cell-fate decisions under stress conditions. Induction of stress granules or loss of DDX3X in the myeloid compartment leads to a decrease in the production of inflammasome-dependent cytokines in vivo. Our findings suggest that macrophages use the availability of DDX3X to interpret stress signals and choose between pro-survival stress granules and pyroptotic ASC specks. Together, our data demonstrate the role of DDX3X in driving NLRP3 inflammasome and stress granule assembly, and suggest a rheostat-like mechanistic paradigm for regulating live-or-die cell-fate decisions under stress conditions.

Interactions between Aspergillus fumigatus and Pulmonary Bacteria: Current State of the Field, New Data, and Future Perspective.

Aspergillus fumigatus and Pseudomonas aeruginosa are central fungal and bacterial members of the pulmonary microbiota. The interactions between A. fumigatus and P. aeruginosa have only just begun to be explored. A balance between inhibitory and stimulatory effects on fungal growth was observed in mixed A. fumigatus-P. aeruginosa cultures. Negative interactions have been seen for homoserine-lactones, pyoverdine and pyochelin resulting from iron starvation and intracellular inhibitory reactive oxidant production. In contrast, several types of positive interactions were recognized. Dirhamnolipids resulted in the production of a thick fungal cell wall, allowing the fungus to resist stress. Phenazines and pyochelin favor iron uptake for the fungus. A. fumigatus is able to use bacterial volatiles to promote its growth. The immune response is also differentially regulated by co-infections.

Fungal ligands released by innate immune effectors promote inflammasome activation during Aspergillus fumigatus infection.

Invasive pulmonary aspergillosis causes substantial mortality in immunocompromised individuals. Recognition of Aspergillus fumigatus by the host immune system leads to activation of the inflammasome, which provides protection against infection. However, regulation of inflammasome activation at the molecular level is poorly understood. Here, we describe two distinct pathways that coordinately control inflammasome activation during A. fumigatus infection. The C-type lectin receptor pathway activates both MAPK and NF-κB signalling, which leads to induction of downstream mediators, such as the transcription factor IRF1, and also primes the inflammasomes. Toll-like receptor signalling through the adaptor molecules MyD88 and TRIF in turn mediates efficient activation of IRF1, which induces IRGB10 expression. IRGB10 targets the fungal cell wall, and the antifungal activity of IRGB10 causes hyphae damage, modifies the A. fumigatus surface and inhibits fungal growth. We also demonstrate that one of the major fungal pathogen-associated molecular patterns, ß-glucan, directly triggers inflammasome assembly. Thus, the concerted activation of both Toll-like receptors and C-type lectin receptors is required for IRF1-mediated IRGB10 regulation, which is a key event governing ligand release and inflammasome activation upon A. fumigatus infection.

ASK Family Kinases Are Required for Optimal NLRP3 Inflammasome Priming.

Activation of the multimeric inflammasome complex leads to inflammatory responses to biotic and abiotic triggers. The inflammasome sensor, Nod-like receptor family pyrin domain containing 3 (NLRP3), is activated by a range of stimuli and is tightly regulated to restrict excessive inflammation. Because NLRP3 responds broadly to cellular insults and regulates cell death similar to the stress-activated apoptosis signal-regulating kinases 1 and 2 (ASK1/2), we hypothesized that ASK1/2 may regulate NLRP3 activity. Although essential for mediating NLRP3 inflammasome activation, ASK1/2 were not required for NLRC4 or absent in melanoma 2 inflammasome activation. ASK1/2 was required for NLRP3 up-regulation after lipopolysaccharide treatment in primary bone marrow-derived macrophages and lung fibroblasts as well as during infection with Burkholderia thailandensis and influenza virus. Consistent with reduced NLRP3 expression in response to B. thailandensis, caspase-1 cleavage and cell death were reduced in infected bone marrow-derived macrophages, and mice lacking ASK1/2 were resistant to Burkholderia intranasal infection. Single knockouts of either ASK1 or ASK2 showed a partial role for both ASK1 and ASK2 in NLRP3 up-regulation in response to lipopolysaccharide or B. thailandensis, but ASK2 was required primarily to mediate lethal pathology during intranasal infection in vivo. Our findings identify the ASK1/2 complex as a regulator of NLRP3 activation and highlight a larger role for ASK2 in lung infection during B. thailandensis infection.

Differential roles of caspase-1 and caspase-11 in infection and inflammation.

Caspase-1, also known as interleukin-1ß (IL-1ß)-converting enzyme (ICE), regulates antimicrobial host defense, tissue repair, tumorigenesis, metabolism and membrane biogenesis. On activation within an inflammasome complex, caspase-1 induces pyroptosis and converts pro-IL-1ß and pro-IL-18 into their biologically active forms. "ICE-/-" or "Casp1-/-" mice generated using 129 embryonic stem cells carry a 129-associated inactivating passenger mutation on the caspase-11 locus, essentially making them deficient in both caspase-1 and caspase-11. The overlapping and unique functions of caspase-1 and caspase-11 are difficult to unravel without additional genetic tools. Here, we generated caspase-1-deficient mouse (Casp1Null) on the C57BL/6 J background that expressed caspase-11. Casp1Null cells did not release IL-1ß and IL-18 in response to NLRC4 activators Salmonella Typhimurium and flagellin, canonical or non-canonical NLRP3 activators LPS and ATP, Escherichia coli, Citrobacter rodentium and transfection of LPS, AIM2 activators Francisella novicida, mouse cytomegalovirus and DNA, and the infectious agents Listeria monocytogenes and Aspergillus fumigatus. We further demonstrated that caspase-1 and caspase-11 differentially contributed to the host defense against A. fumigatus infection and to endotoxemia.