Tryptophan catabolites from microbiota engage aryl hydrocarbon receptor and balance mucosal reactivity via interleukin-22.

Endogenous tryptophan (Trp) metabolites have an important role in mammalian gut immune homeostasis, yet the potential contribution of Trp metabolites from resident microbiota has never been addressed experimentally. Here, we describe a metabolic pathway whereby Trp metabolites from the microbiota balance mucosal reactivity in mice. Switching from sugar to Trp as an energy source (e.g., under conditions of unrestricted Trp availability), highly adaptive lactobacilli are expanded and produce an aryl hydrocarbon receptor (AhR) ligand-indole-3-aldehyde-that contributes to AhR-dependent Il22 transcription. The resulting IL-22-dependent balanced mucosal response allows for survival of mixed microbial communities yet provides colonization resistance to the fungus Candida albicans and mucosal protection from inflammation. Thus, the microbiota-AhR axis might represent an important strategy pursued by coevolutive commensalism for fine tuning host mucosal reactivity contingent on Trp catabolism.

Reversion of a fungal genetic code alteration links proteome instability with genomic and phenotypic diversification.

Many fungi restructured their proteomes through incorporation of serine (Ser) at thousands of protein sites coded by the leucine (Leu) CUG codon. How these fungi survived this potentially lethal genetic code alteration and its relevance for their biology are not understood. Interestingly, the human pathogen Candida albicans maintains variable Ser and Leu incorporation levels at CUG sites, suggesting that this atypical codon assignment flexibility provided an effective mechanism to alter the genetic code. To test this hypothesis, we have engineered C. albicans strains to misincorporate increasing levels of Leu at protein CUG sites. Tolerance to the misincorporations was very high, and one strain accommodated the complete reversion of CUG identity from Ser back to Leu. Increasing levels of Leu misincorporation decreased growth rate, but production of phenotypic diversity on a phenotypic array probing various metabolic networks, drug resistance, and host immune cell responses was impressive. Genome resequencing revealed an increasing number of genotype changes at polymorphic sites compared with the control strain, and 80% of Leu misincorporation resulted in complete loss of heterozygosity in a large region of chromosome V. The data unveil unanticipated links between gene translational fidelity, proteome instability and variability, genome diversification, and adaptive phenotypic diversity. They also explain the high heterozygosity of the C. albicans genome and open the door to produce microorganisms with genetic code alterations for basic and applied research.

IL-22 and IDO1 affect immunity and tolerance to murine and human vaginal candidiasis.

The ability to tolerate Candida albicans, a human commensal of the gastrointestinal tract and vagina, implicates that host defense mechanisms of resistance and tolerance cooperate to limit fungal burden and inflammation at the different body sites. We evaluated resistance and tolerance to the fungus in experimental and human vulvovaginal candidiasis (VVC) as well as in recurrent VVC (RVVC). Resistance and tolerance mechanisms were both activated in murine VVC, involving IL-22 and IL-10-producing regulatory T cells, respectively, with a major contribution by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1). IDO1 was responsible for the production of tolerogenic kynurenines, such that replacement therapy with kynurenines restored immunoprotection to VVC. In humans, two functional genetic variants in IL22 and IDO1 genes were found to be associated with heightened resistance to RVVC, and they correlated with increased local expression of IL-22, IDO1 and kynurenines. Thus, IL-22 and IDO1 are crucial in balancing resistance with tolerance to Candida, their deficiencies are risk factors for RVVC, and targeting tolerance via therapeutic kynurenines may benefit patients with RVVC.

Distinct and complementary roles for Aspergillus fumigatus-specific Tr1 and Foxp3+ regulatory T cells in humans and mice.

Unlike induced Foxp3(+) regulatory T cells (Foxp3(+) iTreg) that have been shown to play an essential role in the development of protective immunity to the ubiquitous mold Aspergillus fumigatus, type-(1)-regulatory T cells (Tr1) cells have, thus far, not been implicated in this process. Here, we evaluated the role of Tr1 cells specific for an epitope derived from the cell wall glucanase Crf-1 of A. fumigatus (Crf-1/p41) in antifungal immunity. We identified Crf-1/p41-specific latent-associated peptide(+) Tr1 cells in healthy humans and mice after vaccination with Crf-1/p41+zymosan. These cells produced high amounts of interleukin (IL)-10 and suppressed the expansion of antigen-specific T cells in vitro and in vivo. In mice, in vivo differentiation of Tr1 cells was dependent on the presence of the aryl hydrocarbon receptor, c-Maf and IL-27. Moreover, in comparison to Tr1 cells, Foxp3(+) iTreg that recognize the same epitope were induced in an interferon gamma-type inflammatory environment and more potently suppressed innate immune cell activities. Overall, our data show that Tr1 cells are involved in the maintenance of antifungal immune homeostasis, and most likely play a distinct, yet complementary, role compared with Foxp3(+) iTreg.

Th17/Treg imbalance in murine cystic fibrosis is linked to indoleamine 2,3-dioxygenase deficiency but corrected by kynurenines.

RATIONALE: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator affect the innate epithelial immune function of the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pathogenic fungi. The appreciation of whether such fungi are primarily responsible for or a consequence of ineffective airway inflammation is important for future therapeutics development. OBJECTIVES: To characterize the impact of the tryptophan/kynurenine pathway on pathogenic airway inflammation preventing effective fungal clearance in CF. METHODS: We studied the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the kynurenine pathway of tryptophan degradation, in human and murine CF, the impact of IDO on lung inflammation and immunity in murine CF, and the potential role of tryptophan catabolism in pathogenesis and therapy of fungus-associated lung inflammation. MEASUREMENTS AND MAIN RESULTS: IDO was defective in murine and human CF. Genetic and transcriptional regulatory mechanisms contributed to dysfunctional IDO activity that, in turn, correlated with imbalanced Th17/Treg-cell responses to Aspergillus fumigatus in murine CF. Treatments enhancing IDO function or preventing pathogenic Th17-cell activation restored protective immunity to the fungus and improved lung inflammation in murine CF. CONCLUSIONS: This study provides a link between tryptophan catabolism and lung immune homeostasis in murine CF, representing a proof-of-concept that targeting pathogenic inflammation via IDO-mimetic drugs may benefit patients with CF.

The danger signal S100B integrates pathogen- and danger-sensing pathways to restrain inflammation.

Humans inhale hundreds of Aspergillus conidia without adverse consequences. Powerful protective mechanisms may ensure prompt control of the pathogen and inflammation. Here we reveal a previously unknown mechanism by which the danger molecule S100B integrates pathogen- and danger-sensing pathways to restrain inflammation. Upon forming complexes with TLR2 ligands, S100B inhibited TLR2 via RAGE, through a paracrine epithelial cells/neutrophil circuit that restrained pathogen-induced inflammation. However, upon binding to nucleic acids, S100B activated intracellular TLRs eventually resolve danger-induced inflammation via transcriptional inhibition of S100B. Thus, the spatiotemporal regulation of TLRs and RAGE by S100B provides evidence for an evolving braking circuit in infection whereby an endogenous danger protects against pathogen-induced inflammation and a pathogen-sensing mechanism resolves danger-induced inflammation.

Dectin-1 Y238X polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient- and donor-dependent mechanisms of antifungal immunity.

The C-type lectin receptor Dectin-1 plays a pivotal role in antifungal immunity. In this study, the recently characterized human DECTIN1 Y238X early stop codon polymorphism leading to diminished Dectin-1 receptor activity was studied in relation to invasive aspergillosis susceptibility and severity in patients receiving hematopoietic stem cell transplantation. We found that the presence of the DECTIN1 Y238X polymorphism in either donors or recipients of hematopoietic stem cell transplantation increased susceptibility to aspergillosis, with the risk being highest when the polymorphism was present simultaneously in both donors and recipients (adjusted hazard ratio = 3.9; P = .005). Functionally, the Y238X polymorphism impaired the production of interferon-γ and interleukin-10 (IL-10), in addition to IL-1ß, IL-6, and IL-17A, by human peripheral mononuclear cells and Dectin-1 on human epithelial cells contributed to fungal recognition. Mechanistically, studies on preclinical models of infection in intact or bone marrow-transplanted Dectin-1 knockout mice revealed that protection from infection requires a distinct, yet complementary, role of both donor and recipient Dectin-1. This study discloses Dectin-1 deficiency as a novel susceptibility factor for aspergillosis in high-risk patients and identifies a previously unsuspected role for Dectin-1 in antifungal immunity that is the ability to control both resistance and tolerance to the fungus contingent on hematopoietic/nonhematopoietic compartmentalization.

Exogenous pentraxin 3 restores antifungal resistance and restrains inflammation in murine chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening bacterial and fungal infections and hyperinflammation. The susceptibility to aspergillosis in experimental CGD (p47(phox-/-) mice) is associated with the failure to control the inherent inflammatory response to the fungus and to restrict the activation of inflammatory Th17 cells. We assessed whether pentraxin (PTX)3, a member of a family of multimeric pattern-recognition proteins with potent anti-Aspergillus activity, could limit pathogenic inflammation in p47(phox-/-) mice by curbing the IL-23/Th17 inflammatory axis in response to the fungus. We found that the production of PTX3 was delayed in CGD mice in infection but exogenous administration of PTX3 early in infection restored antifungal resistance and restrained the inflammatory response to the fungus. This occurred through down-regulation of IL-23 production by dendritic cells and epithelial cells which resulted in limited expansion of IL-23R+ gammadelta+ T cells producing IL-17A and the emergence of Th1/Treg responses with minimum pathology. Thus, PTX3 could be therapeutically used for the exploitation of NADPH-independent mechanism(s) of antifungal immune protection with limited immunopathology in CGD.

Immune sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and the impact on Th immunity and vaccination.

The ability of the fungus Aspergillus fumigatus to activate, suppress, or subvert host immune response during life cycle in vivo through dynamic changing of cell wall structure and secretion implicates discriminative immune sensing of distinct fungal components. In this study, we have comparatively assessed secreted- and membrane-anchored proteins, glycolipids, and polysaccharides for the ability to induce vaccine-dependent protection in transplanted mice and Th cytokine production by human-specific CD4(+) T cell clones. The results show that the different fungal components are endowed with the distinct capacity to activate Th cell responses in mice and humans, with secreted proteins inducing Th2 cell activation, membrane proteins Th1/Treg, glycolipids Th17, and polysaccharides mostly IL-10 production. Of interest, the side-by-side comparison revealed that at least three fungal components (a protease and two glycosylphosphatidylinositol-anchored proteins) retained their immunodominant Th1/Treg activating potential from mice to humans. This suggests that the broadness and specificity of human T cell repertoire against the fungus could be selectively exploited with defined immunoactive Aspergillus Ags.

Protection against Pseudomonas aeruginosa lung infection in mice by recombinant OprF-pulsed dendritic cell immunization.

BACKGROUND: The Pseudomonas aeruginosa major constitutive outer membrane porin protein F (OprF) has been shown to be a protective antigen and was previously used to activate an immunological response in a mouse model of lung pneumonia. The purpose of our study was to demonstrate the ability of mouse dendritic cells pulsed with purified or recombinant OprF to protect mice against P. aeruginosa infection and inflammation.Both native (n-OprF), isolated and purified from PAO1 bacterial strain, and recombinant (histidin-conjugated) OprF (His-OprF), obtained by cloning of the oprF gene into the pET28a expression vector, were used to stimulate dendritic cells in vitro before adoptive transfer into prospective recipient mice with P. aeruginosa pulmonary infection. RESULTS: Similar to n-OprF, His-OprF activated dendritic cells in vitro, inducing the costimulatory molecule expression as well as cytokine production. Upon adoptive transfer in vivo, porin-pulsed dendritic cells (DCs) induced Th1-mediated resistance to infection and associated inflammatory pathology caused by either the PAO1 strain or a clinically-isolated mucoid strain. CONCLUSIONS: This study highlights the pivotal contribution of DCs to vaccine-induced protection against P. aeruginosa infection and associated inflammation.

Interleukin-1 beta levels in gingival crevicular fluid and serum under naturally occurring and experimentally induced gingivitis.

AIMS: To evaluate the interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid (GCF) and serum in either naturally occurring (N-O) or experimentally induced (E-I) plaque-associated gingivitis. MATERIAL AND METHODS: Thirty-seven periodontally healthy subjects were evaluated in real life conditions (N-O gingivitis) as well as after 21 days of experimental gingivitis trial (E-I gingivitis). During the experimental gingivitis trial, in one maxillary quadrant (test quadrant), gingival inflammation was induced by oral hygiene abstention, while in the contralateral (control) quadrant, oral hygiene was routinely continued. IL-1 beta concentrations in N-O and E-I gingivitis were investigated for IL-1B(+3954) and IL-1B(-511) gene polymorphisms. RESULTS: (i) GCF IL-1 beta concentrations in E-I gingivitis were significantly higher compared with N-O gingivitis; (ii) an intra-individual correlation between GCF concentrations of IL-1 beta detected in N-O and E-I gingivitis was observed in control quadrants, but not in test quadrants; (iii) IL-1 beta concentration in GCF was associated with IL-1B(+3954) genotype only at test quadrants; (iv) IL-1 beta was detectable in serum only at low levels in a limited number of subjects, without difference between gingivitis conditions. CONCLUSIONS: Aspects of the bacterial challenge to the gingival tissues, such as the amount of plaque deposits and plaque accumulation rate, appear to affect the IL-1 beta levels in GCF in subjects with a specific IL-1B genotype.

Th17 cells in the setting of Aspergillus infection and pathology.

Innate and adaptive immune responses act to generate the most effective form of immunity for protection against Aspergillus fumigatus. The decision of how to respond is still primarily determined by interactions between fungi and cells of the innate immune system, but the actions of T cells will feed back into this dynamic equilibrium to regulate the balance between pro-inflammatory and anti-inflammatory signals. The enzyme indoleamine 2,3-dioxygenase, and tryptophan metabolites, acting as a bridge between dendritic cells and regulatory T cells, pivotally contribute to such a homeostatic condition by taming inflammatory responses. IL-23 and the newly described Th17 pathway, by means of negative regulation of tryptophan catabolism, play an inflammatory role previously attributed to uncontrolled Th1 response. Our data support a model in which IL-23/IL-17A/Th17-driven inflammation promotes infection and impairs antifungal immune resistance. Thus, modulation of the inflammatory response represents a potential strategy to stimulate protective immune responses to Aspergillus.

Indoleamine 2,3-dioxygenase (IDO) in inflammation and allergy to Aspergillus.

Innate responses combine with adaptive immunity to generate the most effective form of resistance against Aspergillus fumigatus. A complex set of signaling networks initiate both innate and adaptive immunity in response to the different fungal morphotypes. In response, the fungus has developed or acquired sophisticated mechanisms to avoid, counteract and subvert sensors, signaling networks and a range of effector functions that constitute the host immune response. Host response and fungal countermeasures may contribute to the balance of pro-inflammatory and anti-inflammatory signaling that is eventually required to benefit both parties. Here we highlight the important contribution of the enzyme IDO (indoleamine 2,3-dioxygenase) and tryptophan catabolites to such a homeostatic condition in Aspergillus infection and allergy. By providing the host with immune defense mechanisms adequate for protection, without necessarily eliminating the fungus or causing an unacceptable level of tissue damage, IDO and tryptophan metabolites may prove to be potent regulators capable of taming innate and adaptive pathogenic inflammatory host responses.

Inhibition of amniotic interleukin-6 and prostaglandin E2 release by ampicillin.

OBJECTIVE: To test the effect of ampicillin on amniotic interleukin-6 (IL-6) and prostaglandin E2 (PGE2) release. METHODS: In an in vitro study, IL-6 and PGE2 release from amnion-like Wistar Institute Susan Hayflick cells was assayed under basal conditions, as well as after incubation with ampicillin. In an in vivo study, amniotic fluid IL-6 was assayed in a total of 212 patients submitted to genetic amniocentesis during the 17th week of their singleton physiological pregnancy. The study population was subdivided as follows: 92 patients sampled before ampicillin administration, 70 patients sampled 4 hours after administration of 1 g ampicillin, and 50 patients sampled 12 hours after administration of 1 g ampicillin. RESULTS: At doses ranging from 10-7 to 10-4 M, ampicillin decreased IL-6 release from Wistar Institute Susan Hayflick cells. The drug effect was already statistically significant (-30%; P <.05) at the lowest concentration tested (10-7 M), reaching the maximum (-50%) at 10-6 M after 4 hours of incubation. Moreover, ampicillin concentrations ranging from 10-7 to 10-4 M decreased PGE2 release from Wistar Institute Susan Hayflick cells; maximal inhibition was reached at 10-6 M after 4 hours (-40%; P <.05). Finally, IL-6 levels measured in amniotic fluid of patients sampled 4 hours after ampicillin administration proved strongly and significantly reduced when compared with those sampled either before or 12 hours after treatment (P <.001). CONCLUSION: The capacity of ampicillin to directly decrease amniotic IL-6 and PGE2 release should be considered in the management of bacterial and nonbacterial inflammatory complications of pregnancy mediated by the cytokine and prostanoid interaction.

Strain dependent variation of immune responses to A. fumigatus: definition of pathogenic species.

For over a century microbiologists and immunologist have categorized microorganisms as pathogenic or non-pathogenic species or genera. This definition, clearly relevant at the strain and species level for most bacteria, where differences in virulence between strains of a particular species are well known, has never been probed at the strain level in fungal species. Here, we tested the immune reactivity and the pathogenic potential of a collection of strains from Aspergillus spp, a fungus that is generally considered pathogenic in immuno-compromised hosts. Our results show a wide strain-dependent variation of the immune response elicited indicating that different isolates possess diverse virulence and infectivity. Thus, the definition of markers of inflammation or pathogenicity cannot be generalized. The profound understanding of the molecular mechanisms subtending the different immune responses will result solely from the comparative study of strains with extremely diverse properties.

Inflammation in aspergillosis: the good, the bad, and the therapeutic.

Aspergillosis includes a spectrum of diseases caused by different Aspergillus spp. New insights into the cellular and molecular mechanisms of resistance and immune tolerance to the fungus in infection and allergy have been obtained in experimental settings. The fact that virulence factors, traditionally viewed as fungal attributes, are contingent upon microbial adaptation to various environmental stresses encountered in the human host implies that the host and fungus are jointly responsible for pathogenicity. Ultimately, despite the occurrence of severe aspergillosis in immunocompromised patients, clinical evidence indicates that aspergillosis also occurs in the setting of a heightened inflammatory response, in which immunity occurs at the expense of host damage and pathogen eradication. Thus, targeting pathogenicity rather than microbial growth, tolerance rather than resistance mechanisms of defense may pave the way to targeted anti-inflammatory strategies in difficult-to-treat patients. The challenge now is to translate promising results from experimental models to the clinic.

Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis.

The recognition of ß-glucans by dectin-1 has been shown to mediate cell activation, cytokine production and a variety of antifungal responses. Here, we report that the functional activity of dectin-1 in mucosal immunity to Candida albicans is influenced by the genetic background of the host. Dectin-1 was required for the proper control of gastrointestinal and vaginal candidiasis in C57BL/6, but not BALB/c mice; in fact, the latter showed increased resistance in the absence of dectin-1. The susceptibility of dectin-1-deficient C57BL/6 mice to infection was associated with defects in IL-17A and aryl hydrocarbon receptor-dependent IL-22 production and in adaptive Th1 responses. In contrast, the resistance of dectin-1-deficient BALB/c mice was associated with increased IL-17A and IL-22 production and the skewing towards Th1/Treg immune responses that provide immunological memory. Disparate canonical/noncanonical NF-κB signaling pathways downstream of dectin-1 were activated in the two different mouse strains. Thus, the net activity of dectin-1 in antifungal mucosal immunity is dependent on the host's genetic background, which affects both the innate cytokine production and the adaptive Th1/Th17 cell activation upon dectin-1 signaling.

CD4(+) T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease.

Aspergillus fumigatus is a model fungal pathogen and a common cause of infection in individuals with the primary immunodeficiency chronic granulomatous disease (CGD). Although primarily considered a deficiency of innate immunity, CGD is also linked to dysfunctional T cell reactivity. Both CD4(+) and CD8(+) T cells mediate vaccine-induced protection from experimental aspergillosis, but the molecular mechanisms leading to the generation of protective immunity and whether these mechanisms are dysregulated in individuals with CGD have not been determined. Here, we show that activation of either T cell subset in a mouse model of CGD is contingent upon the nature of the fungal vaccine, the involvement of distinct innate receptor signaling pathways, and the mode of antigen routing and presentation in DCs. Aspergillus conidia activated CD8(+) T cells upon sorting to the Rab14(+) endosomal compartment required for alternative MHC class I presentation. Cross-priming of CD8(+) T cells failed to occur in mice with CGD due to defective DC endosomal alkalinization and autophagy. However, long-lasting antifungal protection and disease control were successfully achieved upon vaccination with purified fungal antigens that activated CD4(+) T cells through the endosome/lysosome pathway. Our study thus indicates that distinct intracellular pathways are exploited for the priming of CD4(+) and CD8(+) T cells to A. fumigatus and suggests that CD4(+) T cell vaccination may be able to overcome defective antifungal CD8(+) T cell memory in individuals with CGD.

Genetically-determined hyperfunction of the S100B/RAGE axis is a risk factor for aspergillosis in stem cell transplant recipients.

Invasive aspergillosis (IA) is a major threat to the successful outcome of hematopoietic stem cell transplantation (HSCT), although individual risk varies considerably. Recent evidence has established a pivotal role for a danger sensing mechanism implicating the S100B/receptor for advanced glycation end products (RAGE) axis in antifungal immunity. The association of selected genetic variants in the S100B/RAGE axis with susceptibility to IA was investigated in 223 consecutive patients undergoing HSCT. Furthermore, studies addressing the functional consequences of these variants were performed. Susceptibility to IA was significantly associated with two distinct polymorphisms in RAGE (-374T/A) and S100B (+427C/T) genes, the relative contribution of each depended on their presence in both transplantation counterparts [patient SNP(RAGE), adjusted hazard ratio (HR), 1.97; P = 0.042 and donor SNP(RAGE), HR, 2.03; P = 0.047] or in donors (SNP(S100B), HR, 3.15; P = 7.8e-(4)) only, respectively. Functional assays demonstrated a gain-of-function phenotype of both variants, as shown by the enhanced expression of inflammatory cytokines in RAGE polymorphic cells and increased S100B secretion in vitro and in vivo in the presence of the S100B polymorphism. These findings point to a relevant role of the danger sensing signaling in human antifungal immunity and highlight a possible contribution of a genetically-determined hyperfunction of the S100B/RAGE axis to susceptibility to IA in the HSCT setting.

Lack of Toll IL-1R8 exacerbates Th17 cell responses in fungal infection.

TLRs contribute to the inflammatory response in fungal infections. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. In this study, we tested the hypothesis that Toll IL-1R8 (TIR8)/single Ig IL-1-related receptor, a member of the IL-1R family acting as a negative regulator of TLR/IL-1R signaling, affects TLR responses in fungal infections. Genetically engineered Tir8(-/-) mice were assessed for inflammatory and adaptive Th cell responses to Candida albicans and Aspergillus fumigatus. Inflammatory pathology and susceptibility to infection were higher in Tir8(-/-) mice and were causally linked to the activation of the Th17 pathway. IL-1R signaling was involved in Th17 cell activation by IL-6 and TGF-beta in that limited inflammatory pathology and relative absence of Th17 cell activation were observed in IL-1RI(-/-) mice. These data demonstrate that TIR8 is required for host resistance to fungal infections and that it functions to negatively regulate IL-1-dependent activation of inflammatory Th17 responses. TIR8 may contribute toward fine-tuning the balance between protective immunity and immunopathology in infection.