Large-scale mutational analysis identifies UNC93B1 variants that drive TLR-mediated autoimmunity in mice and humans.

Nucleic acid-sensing Toll-like receptors (TLR) 3, 7/8, and 9 are key innate immune sensors whose activities must be tightly regulated to prevent systemic autoimmune or autoinflammatory disease or virus-associated immunopathology. Here, we report a systematic scanning-alanine mutagenesis screen of all cytosolic and luminal residues of the TLR chaperone protein UNC93B1, which identified both negative and positive regulatory regions affecting TLR3, TLR7, and TLR9 responses. We subsequently identified two families harboring heterozygous coding mutations in UNC93B1, UNC93B1+/T93I and UNC93B1+/R336C, both in key negative regulatory regions identified in our screen. These patients presented with cutaneous tumid lupus and juvenile idiopathic arthritis plus neuroinflammatory disease, respectively. Disruption of UNC93B1-mediated regulation by these mutations led to enhanced TLR7/8 responses, and both variants resulted in systemic autoimmune or inflammatory disease when introduced into mice via genome editing. Altogether, our results implicate the UNC93B1-TLR7/8 axis in human monogenic autoimmune diseases and provide a functional resource to assess the impact of yet-to-be-reported UNC93B1 mutations.

Disrupted degradative sorting of TLR7 is associated with human lupus.

Hyperactive TLR7 signaling has long been appreciated as driver of autoimmune disease in mouse models. Recently, gain-of-function mutations in TLR7 were identified as a monogenic cause of human lupus. TLR7 is an intracellular transmembrane receptor, sensing RNA breakdown products within late endosomes. Here, we show that endosome dysfunction leads to unrestricted TLR7 signaling and is associated with human lupus. The late endosomal BORC complex together with the small GTPase Arl8b controls intracellular TLR7 levels by regulating receptor turnover. This requires a direct interaction between the TLR7-associated trafficking factor Unc93b1 and Arl8b. We identified an UNC93B1 mutation in a patient with childhood-onset lupus, which results in reduced BORC interaction and endosomal TLR7 accumulation. Therefore, a failure to control TLR7 turnover is sufficient to break immunological tolerance to nucleic acids. Our results highlight the importance of an intact endomembrane system in preventing pathological TLR7 signaling and autoimmune disease.

UNC93B1 variants underlie TLR7-dependent autoimmunity.

UNC93B1 is critical for trafficking and function of nucleic acid-sensing Toll-like receptors (TLRs) TLR3, TLR7, TLR8, and TLR9, which are essential for antiviral immunity. Overactive TLR7 signaling induced by recognition of self-nucleic acids has been implicated in systemic lupus erythematosus (SLE). Here, we report UNC93B1 variants (E92G and R336L) in four patients with early-onset SLE. Patient cells or mouse macrophages carrying the UNC93B1 variants produced high amounts of TNF-α and IL-6 and upon stimulation with TLR7/TLR8 agonist, but not with TLR3 or TLR9 agonists. E92G causes UNC93B1 protein instability and reduced interaction with TLR7, leading to selective TLR7 hyperactivation with constitutive type I IFN signaling. Thus, UNC93B1 regulates TLR subtype-specific mechanisms of ligand recognition. Our findings establish a pivotal role for UNC93B1 in TLR7-dependent autoimmunity and highlight the therapeutic potential of targeting TLR7 in SLE.

UNC93B1 recruits syntenin-1 to dampen TLR7 signalling and prevent autoimmunity.

At least two members of the Toll-like receptor (TLR) family, TLR7 and TLR9, can recognize self-RNA and self-DNA, respectively. Despite the structural and functional similarities between these receptors, their contributions to autoimmune diseases such as systemic lupus erythematosus can differ. For example, TLR7 and TLR9 have opposing effects in mouse models of systemic lupus erythematosus-disease is exacerbated in TLR9-deficient mice but attenuated in TLR7-deficient mice1. However, the mechanisms of negative regulation that differentiate between TLR7 and TLR9 are unknown. Here we report a function for the TLR trafficking chaperone UNC93B1 that specifically limits signalling of TLR7, but not TLR9, and prevents TLR7-dependent autoimmunity in mice. Mutations in UNC93B1 that lead to enhanced TLR7 signalling also disrupt binding of UNC93B1 to syntenin-1, which has been implicated in the biogenesis of exosomes2. Both UNC93B1 and TLR7 can be detected in exosomes, suggesting that recruitment of syntenin-1 by UNC93B1 facilitates the sorting of TLR7 into intralumenal vesicles of multivesicular bodies, which terminates signalling. Binding of syntenin-1 requires phosphorylation of UNC93B1 and provides a mechanism for dynamic regulation of TLR7 activation and signalling. Thus, UNC93B1 not only enables the proper trafficking of nucleic acid-sensing TLRs, but also sets the activation threshold of potentially self-reactive TLR7.

Release from UNC93B1 reinforces the compartmentalized activation of select TLRs.

Nucleic acid-sensing Toll-like receptors (TLRs) are subject to complex regulation to facilitate the recognition of microbial DNA and RNA while limiting the recognition of an organism's own nucleic acids1. Failure to properly regulate these TLRs can lead to autoimmune and autoinflammatory diseases2-6. Intracellular localization of these receptors is thought to be crucial for the discrimination between self and non-self7, but the molecular mechanisms that reinforce compartmentalized activation of intracellular TLRs remain poorly understood. Here we describe a mechanism that prevents the activation of TLR9 from locations other than endosomes. This control is achieved through the regulated release of the receptor from its trafficking chaperone UNC93B1, which occurs only within endosomes and is required for ligand binding and signal transduction. Preventing release of TLR9 from UNC93B1, either by mutations in UNC93B1 that increase affinity for TLR9 or through an artificial tether that impairs release, results in defective signalling. Whereas TLR9 and TLR3 are released from UNC93B1, TLR7 does not dissociate from UNC93B1 in endosomes and is regulated by distinct mechanisms. This work defines a checkpoint that reinforces the compartmentalized activation of TLR9, and provides a mechanism by which activation of individual endosomal TLRs may be distinctly regulated.

Nucleic acid-sensing TLRs: trafficking and regulation.

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-derived and host-derived nucleic acid ligands. 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.

The tyrosine kinase Btk regulates the macrophage response to Listeria monocytogenes infection.

In this study we investigated the role of Bruton's tyrosine kinase (Btk) in the immune response to the Gram-positive intracellular bacterium Listeria monocytogenes (Lm). In response to Lm infection, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk (-/-) BMMs showed enhanced TNF-α, IL-6 and IL-12p40 secretion, while type I interferons were produced at levels similar to wild-type (wt) BMMs. Although Btk-deficient BMMs displayed reduced phagocytosis of E. coli fragments, there was no difference between wt and Btk (-/-) BMMs in the uptake of Lm upon infection. Moreover, there was no difference in the response to heat-killed Lm between wt and Btk (-/-) BMMs, suggesting a role for Btk in signaling pathways that are induced by intracellular Lm. Finally, Btk (-/-) mice displayed enhanced resistance and an increased mean survival time upon Lm infection in comparison to wt mice. This correlated with elevated IFN-γ and IL-12p70 serum levels in Btk (-/-) mice at day 1 after infection. Taken together, our data suggest an important regulatory role for Btk in macrophages during Lm infection.

Type I interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during Candida infections.

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 Ly6C(hi) 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.

Transcription factor Efg1 shows a haploinsufficiency phenotype in modulating the cell wall architecture and immunogenicity of Candida albicans.

The Candida albicans transcription factor Efg1 is known to be involved in many different cellular processes, including morphogenesis, general metabolism, and virulence. Here we show that besides its manifold roles, Efg1 also has a prominent effect on cell wall structure and composition, strongly affecting the structural glucan part. Deletion of only one allele of EFG1 already results in severe phenotypes for cell wall biogenesis, comparable to those with deletion of both alleles, indicative of a severe haploinsufficiency for EFG1. The observed defects in structural setup of the cell wall, together with previously reported alterations in expression of cell surface proteins, result in altered immunogenic properties of strains with compromised Efg1 function. This is shown by interaction studies with macrophages and primary dendritic cells. The structural changes in the cell wall carbohydrate meshwork presented here, together with the manifold changes in cell wall protein composition and metabolism reported in other studies, contribute to the altered immune response mounted by innate immune cells and to the altered virulence phenotypes observed for strains lacking EFG1.

The facultative intracellular pathogen Candida glabrata subverts macrophage cytokine production and phagolysosome maturation.

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.

Conventional dendritic cells mount a type I IFN response against Candida spp. requiring novel phagosomal TLR7-mediated IFN-ß signaling.

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-ß response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/ß receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-ß release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-ß and the ß-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-ß 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-ß 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.

Fungal attacks on mammalian hosts: pathogen elimination requires sensing and tasting.

Recognition of Candida spp. by immune cells is mediated by dedicated pattern recognition receptors (PRRs), including Toll-like receptors (TLRs) and lectins expressed on innate immune cells (e.g., macrophages, neutrophils and dendritic cells (DCs)). PRRs recognize Candida-specific pathogen-associated molecular patterns (PAMPs). Binding of fungal PAMPs (e.g., cell wall sugar polymers and proteins, fungal nucleic acids) to PRRs triggers the activation of innate effector cells. Recent findings underscore the role of DCs in relaying PAMP information through their PRRs to stimulate the adaptive response. In agreement, deficiencies in certain PRRs strongly impair survival to Candida infections in mice and is associated with enhanced susceptibility to mucocutaneous fungal infections in humans. Understanding the complex signaling networks protecting the host against fungal pathogens remains a challenge in the field.

The Set3/Hos2 histone deacetylase complex attenuates cAMP/PKA signaling to regulate morphogenesis and virulence of Candida albicans.

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 set3Delta/Delta 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.

In vitro systems for studying the interaction of fungal pathogens with primary cells from the mammalian innate immune system.

The incidence of invasive fungal diseases has increased over the past decades, particularly in relation with the increase of immunocompromised patient cohorts (e.g., HIV-infected patients, transplant recipients, immunosuppressed patients with cancer). Opportunistic fungal pathogens such as Candida spp. are most often associated with serious systemic infections. Currently available antifungal drugs are rather unspecific, often with severe side effects. In some cases, their prophylactic use has favored emergence of resistant fungal strains. Major antifungal drugs target the biosynthesis of lipid components of the fungal plasma membrane or the assembly of the cell wall. For a more specific and efficient treatment and prevention of fungal infection, new therapeutic strategies are needed, including strengthening or stimulation of the residual host immune response. Achieving such a goal requires a better understanding of factors important for the defense and the survival of the host combating Candida spp. Where possible, primary cultures of mammalian immune cells of the innate immune system constitute a better suited model than transformed cell lines to study host-pathogen response and virulence. Hence, in vitro primary cell culture systems are a good strategy for a first screening of mutant strains of Candida spp. to identify virulence traits with regard to host cell response and pathogen invasion.

Candida albicans cell surface superoxide dismutases degrade host-derived reactive oxygen species to escape innate immune surveillance.

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 sod5Delta/Delta and sod4Delta/Deltasod5Delta/Delta 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.