Mammalian innate immune cells produce reactive oxygen species (ROS) in the oxidative burst reaction to destroy invading microbial pathogens. Using quantitative real-time ROS assays, we show here that both yeast and filamentous forms of the opportunistic human fungal pathogen Candida albicans trigger ROS production in primary innate immune cells such as macrophages and dendritic cells. Through a reverse genetic approach, we demonstrate that coculture of macrophages or myeloid dendritic cells with C. albicans cells lacking the superoxide dismutase (SOD) Sod5 leads to massive extracellular ROS accumulation in vitro. ROS accumulation was further increased in coculture with fungal cells devoid of both Sod4 and Sod5. Survival experiments show that C. albicans mutants lacking Sod5 and Sod4 exhibit a severe loss of viability in the presence of macrophages in vitro. The reduced viability of sod5Δ/Δ and sod4Δ/Δsod5Δ/Δ mutants relative to wild type is not evident with macrophages from gp91phox−/− mice defective in the oxidative burst activity, demonstrating a ROS-dependent killing activity of macrophages targeting fungal pathogens. These data show a physiological role for cell surface SODs in detoxifying ROS, and suggest a mechanism whereby C. albicans, and perhaps many other microbial pathogens, can evade host immune surveillance in vivo.

Although Candida glabrata is an important human pathogenic yeast, its pathogenicity mechanisms are largely unknown. Immune evasion strategies seem to play key roles during infection, since very little inflammation is observed in mouse models. Furthermore, C. glabrata multiplies intracellularly after engulfment by macrophages. In this study, we sought to identify the strategies that enable C. glabrata to survive phagosome biogenesis and antimicrobial activities within human monocyte-derived macrophages. We show that, despite significant intracellular proliferation, macrophage damage or apoptosis was not apparent, and production of reactive oxygen species was inhibited. Additionally, with the exception of GM-CSF, levels of pro- and anti-inflammatory cytokines were only marginally increased. We demonstrate that adhesion to and internalization by macrophages occur within minutes, and recruitment of endosomal early endosomal Ag 1 and lysosomal-associated membrane protein 1 indicates phagosome maturation. However, phagosomes containing viable C. glabrata, but not heat-killed yeasts, failed to recruit cathepsin D and were only weakly acidified. This inhibition of acidification did not require fungal viability, but it had a heat-sensitive surface attribute. Therefore, C. glabrata modifies the phagosome into a nonacidified environment and multiplies until the host cells finally lyse and release the fungi. Our results suggest persistence of C. glabrata within macrophages as a possible immune evasion strategy.

Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1−/−), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6Chi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1−/− mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.

Candida albicans, like other pleiomorphic fungal pathogens, is able to undergo a reversible transition between single yeast-like cells and multicellular filaments. This morphogenetic process has long been considered as a key fungal virulence factor. Here, we identify the evolutionarily conserved Set3/Hos2 histone deacetylase complex (Set3C) as a crucial repressor of the yeast-to-filament transition. Cells lacking core components of the Set3C are able to maintain all developmental phases, but are hypersusceptible to filamentation-inducing signals, because of a hyperactive cAMP/Protein Kinase A signaling pathway. Strikingly, Set3C-mediated control of filamentation is required for virulence in vivo, since set3Δ/Δ cells display strongly attenuated virulence in a mouse model of systemic infection. Importantly, the inhibition of histone deacetylase activity by trichostatin A exclusively phenocopies the absence of a functional Set3C, but not of any other histone deacetylase gene. Hence, our work supports a paradigm for manipulating morphogenesis in C. albicans through alternative antifungal therapeutic strategies.

At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA or DNA, respectively. Despite the structural and functional similarities between these receptors, their contribution to autoimmune diseases such as systemic lupus erythematosus (SLE) can be quite different. For example, TLR7 and TLR9 have opposing effects in mouse models of SLE; disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice 1 . However, mechanisms of negative regulation that differentiate between TLR7 and TLR9 have not been described. Here we report a new function for the TLR trafficking chaperone Unc93b1 that specifically limits signaling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. We find that mutations in Unc93b1 leading to enhanced TLR7 signaling also disrupt binding to Syntenin-1, a protein implicated in exosome biogenesis. Both Unc93b1 and TLR7 are detectable in exosomes, suggesting that Unc93b1 recruitment of Syntenin-1 facilitates sorting of TLR7 into intralumenal vesicles of multivesicular bodies which terminates signaling. Syntenin-1 binding requires phosphorylation of Unc93b1, providing a mechanism for dynamic regulation of TLR7 activation and signaling. Thus, Unc93b1 not only enables proper trafficking of nucleic acidsensing TLRs but also sets the activation threshold of potentially self-reactive TLR7.Recognition of nucleic acids enables detection of diverse pathogens by a limited number of innate immune receptors but also exposes the host to potential autoimmunity 2,3 . Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-β response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/β receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-β release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-β and the β-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-β release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-β signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata.

Toll-like receptors (TLRs) play an important role in innate immune responses against pathogenic microorganisms or tissue damage. Nucleic acid (NA)-sensing TLRs localize in intracellular vesicular compartments and recognize foreign- and host-derived nucleotides. Inappropriate activation of NA-sensing TLRs can cause pathogenic inflammation and autoimmunity. Multiple regulatory mechanisms exist to limit recognition of self-NAs. This review summarizes recent progress that has been made in understanding how NA-sensing TLRs are regulated via trafficking, proteolytic cleavage, as well as ligand processing and recognition.

Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate recognition of microbial DNA and RNA while limiting recognition of self-nucleic acids 1 . Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory disease [2][3][4][5][6] . Intracellular localization of these receptors is thought to be critical for self vs. non-self discrimination 7 , yet the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a new mechanism that prevents TLR9 activation from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone Unc93b1, which only occurs within endosomes and is required for ligand binding and signal transduction. Preventing TLR9 release from Unc93b1, either through mutations in Unc93b1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signaling. While TLR9 and TLR3 release from Unc93b1, TLR7 does not dissociate from Unc93b1 in endosomes and is regulated via distinct mechanisms. This work defines a new checkpoint that reinforces compartmentalized activation of TLR9 and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated.The multi-pass transmembrane protein Unc93b1 contributes to the compartmentalized activation of nucleic acid-sensing TLRs by mediating their trafficking from the ER to endosomes 8 . Mutations in Unc93b1 that result in aberrant TLR trafficking can lead to autoimmune disease 3,9 . It has been suggested that Unc93b1 may regulate additional sorting steps that are distinct between individual TLRs 10 ; however, the mechanisms by which Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: