Amniotic fluid stem cell-derived extracellular vesicles educate type 2 conventional dendritic cells to rescue autoimmune disorders in a multiple sclerosis mouse model.

Dendritic cells (DCs) are essential orchestrators of immune responses and represent potential targets for immunomodulation in autoimmune diseases. Human amniotic fluid secretome is abundant in immunoregulatory factors, with extracellular vesicles (EVs) being a significant component. However, the impact of these EVs on dendritic cells subsets remain unexplored. In this study, we investigated the interaction between highly purified dendritic cell subsets and EVs derived from amniotic fluid stem cell lines (HAFSC-EVs). Our results suggest that HAFSC-EVs are preferentially taken up by conventional dendritic cell type 2 (cDC2) through CD29 receptor-mediated internalization, resulting in a tolerogenic DC phenotype characterized by reduced expression and production of pro-inflammatory mediators. Furthermore, treatment of cDC2 cells with HAFSC-EVs in coculture systems resulted in a higher proportion of T cells expressing the regulatory T cell marker Foxp3 compared to vehicle-treated control cells. Moreover, transfer of HAFSC-EV-treated cDC2s into an EAE mouse model resulted in the suppression of autoimmune responses and clinical improvement. These results suggest that HAFSC-EVs may serve as a promising tool for reprogramming inflammatory cDC2s towards a tolerogenic phenotype and for controlling autoimmune responses in the central nervous system, representing a potential platform for the study of the effects of EVs in DC subsets.

A microbially produced AhR ligand promotes a Tph1-driven tolerogenic program in multiple sclerosis.

Multiple sclerosis is a debilitating autoimmune disease, characterized by chronic inflammation of the central nervous system. While the significance of the gut microbiome on multiple sclerosis pathogenesis is established, the underlining mechanisms are unknown. We found that serum levels of the microbial postbiotic tryptophan metabolite indole-3-carboxaldehyde (3-IAld) inversely correlated with disease duration in multiple sclerosis patients. Much like the host-derived tryptophan derivative L-Kynurenine, 3-IAld would bind and activate the Aryl hydrocarbon Receptor (AhR), which, in turn, controls endogenous tryptophan catabolic pathways. As a result, in peripheral lymph nodes, microbial 3-IAld, affected mast-cell tryptophan metabolism, forcing mast cells to produce serotonin via Tph1. We thus propose a protective role for AhR-mast-cell activation driven by the microbiome, whereby natural metabolites or postbiotics will have a physiological role in immune homeostasis and may act as therapeutic targets in autoimmune diseases.

Dual species sphingosine-1-phosphate lyase inhibitors to combine antifungal and anti-inflammatory activities in cystic fibrosis: a feasibility study.

Cystic fibrosis (CF) is an autosomal recessive disorder characterized by respiratory failure due to a vicious cycle of defective Cystic Fibrosis Transmembrane conductance Regulator (CFTR) function, chronic inflammation and recurrent bacterial and fungal infections. Although the recent introduction of CFTR correctors/potentiators has revolutionized the clinical management of CF patients, resurgence of inflammation and persistence of pathogens still posit a major concern and should be targeted contextually. On the background of a network-based selectivity that allows to target the same enzyme in the host and microbes with different outcomes, we focused on sphingosine-1-phosphate (S1P) lyase (SPL) of the sphingolipid metabolism as a potential candidate to uniquely induce anti-inflammatory and antifungal activities in CF. As a feasibility study, herein we show that interfering with S1P metabolism improved the immune response in a murine model of CF with aspergillosis while preventing germination of Aspergillus fumigatus conidia. In addition, in an early drug discovery process, we purified human and A. fumigatus SPL, characterized their biochemical and structural properties, and performed an in silico screening to identify potential dual species SPL inhibitors. We identified two hits behaving as competitive inhibitors of pathogen and host SPL, thus paving the way for hit-to-lead and translational studies for the development of drug candidates capable of restraining fungal growth and increasing antifungal resistance.

Activation of TLRs by Opportunistic Fungi in Lung Organoids.

Organoid cultures may express several types of pattern-recognition receptors and in particular toll-like receptors, representing an extremely efficient and innovative system to understand how pathogen-associated molecular patterns exposure may affect the immunity, the growth, or differentiation of complex tissues. Here, we describe how to generate lung organoids from human-induced pluripotent stem cells. Three-dimensional (3D) cultures are then stimulated with different toll-like receptor ligands derived from fungi or with Aspergillus fumigatus. RNA sequencing may be performed upon organoid cultures to understand host-pathogen innate immune interactions.

Microbiota-Associated HAF-EVs Regulate Monocytes by Triggering or Inhibiting Inflammasome Activation.

In pregnancy, human amniotic fluid extracellular vesicles (HAF-EVs) exert anti-inflammatory effects on T cells and on monocytes, supporting their immunoregulatory roles. The specific mechanisms are still not completely defined. The aim of this study was to investigate the ability of HAF-EVs, isolated from pregnant women who underwent amniocentesis and purified by gradient ultracentrifugation, to affect inflammasome activation in the human monocytes. Proteomic studies revealed that HAF-EV samples expressed several immunoregulatory molecules as well as small amounts of endotoxin. Surprisingly, metagenomic analysis shows the presence of specific bacterial strain variants associated with HAF-EVs as potential sources of the endotoxin. Remarkably, we showed that a single treatment of THP-1 cells with HAF-EVs triggered inflammasome activation, whereas the same treatment followed by LPS and ATP sensitization prevented inflammasome activation, a pathway resembling monocyte refractories. A bioinformatics analysis of microbiota-HAF-EVs functional pathways confirmed the presence of enzymes for endotoxin biosynthesis as well as others associated with immunoregulatory functions. Overall, these data suggest that HAF-EVs could serve as a source of the isolation of a specific microbiota during early pregnancy. Moreover, HAF-EVs could act as a novel system to balance immune training and tolerance by modulating the inflammasome in monocytes or other cells.

Lung Organoids-The Ultimate Tool to Dissect Pulmonary Diseases?

Organoids are complex multicellular three-dimensional (3D) in vitro models that are designed to allow accurate studies of the molecular processes and pathologies of human organs. Organoids can be derived from a variety of cell types, such as human primary progenitor cells, pluripotent stem cells, or tumor-derived cells and can be co-cultured with immune or microbial cells to further mimic the tissue niche. Here, we focus on the development of 3D lung organoids and their use as disease models and drug screening tools. We introduce the various experimental approaches used to model complex human diseases and analyze their advantages and disadvantages. We also discuss validation of the organoids and their physiological relevance to the study of lung diseases. Furthermore, we summarize the current use of lung organoids as models of host-pathogen interactions and human lung diseases such as cystic fibrosis, chronic obstructive pulmonary disease, or SARS-CoV-2 infection. Moreover, we discuss the use of lung organoids derived from tumor cells as lung cancer models and their application in personalized cancer medicine research. Finally, we outline the future of research in the field of human induced pluripotent stem cell-derived organoids.

Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Crystal structure of Aspergillus fumigatus AroH, an aromatic amino acid aminotransferase.

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5'-phosphate-dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L-kynurenine and α-aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus-related diseases. We have solved the crystal structure of Af-AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH.

Interleukin-17 affects synaptic plasticity and cognition in an experimental model of multiple sclerosis.

Cognitive impairment (CI) is a disabling concomitant of multiple sclerosis (MS) with a complex and controversial pathogenesis. The cytokine interleukin-17A (IL-17A) is involved in the immune pathogenesis of MS, but its possible effects on synaptic function and cognition are still largely unexplored. In this study, we show that the IL-17A receptor (IL-17RA) is highly expressed by hippocampal neurons in the CA1 area and that exposure to IL-17A dose-dependently disrupts hippocampal long-term potentiation (LTP) through the activation of its receptor and p38 mitogen-activated protein kinase (MAPK). During experimental autoimmune encephalomyelitis (EAE), IL-17A overexpression is paralleled by hippocampal LTP dysfunction. An in vivo behavioral analysis shows that visuo-spatial learning abilities are preserved when EAE is induced in mice lacking IL-17A. Overall, this study suggests a key role for the IL-17 axis in the neuro-immune cross-talk occurring in the hippocampal CA1 area and its potential involvement in synaptic dysfunction and MS-related CI.

Breakdown of Symbiosis in Radiation-Induced Oral Mucositis.

Oral mucositis is an acute side effect of radiation therapy that is especially common with head and neck cancer treatment. In recent years, several studies have revealed the predisposing factors for mucositis, leading to the pre-treatment of patients to deter the development of opportunistic oral fungal infections. Although many clinical protocols already advise the use of probiotics to counteract inflammation and fungal colonization, preclinical studies are needed to better delineate the mechanisms by which a host may acquire benefits via co-evolution with oral microbiota, probiotics, and fungal commensals, such as Candida albicans, especially during acute inflammation. Here, we review the current understanding of radiation therapy-dependent oral mucositis in terms of pathology, prevention, treatment, and related opportunistic infections, with a final focus on the oral microbiome and how it may be important for future therapy.

Pharyngeal Microbial Signatures Are Predictive of the Risk of Fungal Pneumonia in Hematologic Patients.

The ability to predict invasive fungal infections (IFI) in patients with hematological malignancies is fundamental for successful therapy. Although gut dysbiosis is known to occur in hematological patients, whether airway dysbiosis also contributes to the risk of IFI has not been investigated. Nasal and oropharyngeal swabs were collected for functional microbiota characterization in 173 patients with hematological malignancies recruited in a multicenter, prospective, observational study and stratified according to the risk of developing IFI. A lower microbial richness and evenness were found in the pharyngeal microbiota of high-risk patients that were associated with a distinct taxonomic and metabolic profile. A murine model of IFI provided biologic plausibility for the finding that loss of protective anaerobes, such as Clostridiales and Bacteroidetes, along with an apparent restricted availability of tryptophan, is causally linked to the risk of IFI in hematologic patients and indicates avenues for antimicrobial stewardship and metabolic reequilibrium in IFI.

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity.

Indoleamine 2,3-dioxygenases (IDOs) degrade l-tryptophan to kynurenines and drive the de novo synthesis of nicotinamide adenine dinucleotide. Unsurprisingly, various invertebrates, vertebrates, and even fungi produce IDO. In mammals, IDO1 also serves as a homeostatic regulator, modulating immune response to infection via local tryptophan deprivation, active catabolite production, and non-enzymatic cell signaling. Whether fungal Idos have pleiotropic functions that impact on host-fungal physiology is unclear. Here, we show that Aspergillus fumigatus possesses three ido genes that are expressed under conditions of hypoxia or tryptophan abundance. Loss of these genes results in increased fungal pathogenicity and inflammation in a mouse model of aspergillosis, driven by an alternative tryptophan degradation pathway to indole derivatives and the host aryl hydrocarbon receptor. Fungal tryptophan metabolic pathways thus cooperate with the host xenobiotic response to shape host-microbe interactions in local tissue microenvironments.

Regulation of host physiology and immunity by microbial indole-3-aldehyde.

Co-evolution of the microbial communities with the mammalian host has resulted in intertwined metabolic pathways ultimately affecting physiological and pathological processes. Tryptophan derivatives of host and microbial origin are emblematic of this metabolic promiscuity. One such metabolite, indole-3-aldehyde (3-IAld), is produced by the gut microbiota and was originally identified for its ability to promote epithelial barrier functions by working as an agonist of the Aryl hydrocarbon Receptor. This original observation has been extended in the recent years to include a plethora of activities in several pathological conditions. In this review, we describe the multifaceted role of 3-IAld in host physiology, pathology and immunity and discuss how its proper clinical development may turn into a valuable therapeutic strategy.

Pyridoxal 5'-Phosphate-Dependent Enzymes at the Crossroads of Host-Microbe Tryptophan Metabolism.

The chemical processes taking place in humans intersects the myriad of metabolic pathways occurring in commensal microorganisms that colonize the body to generate a complex biochemical network that regulates multiple aspects of human life. The role of tryptophan (Trp) metabolism at the intersection between the host and microbes is increasingly being recognized, and multiple pathways of Trp utilization in either direction have been identified with the production of a wide range of bioactive products. It comes that a dysregulation of Trp metabolism in either the host or the microbes may unbalance the production of metabolites with potential pathological consequences. The ability to redirect the Trp flux to restore a homeostatic production of Trp metabolites may represent a valid therapeutic strategy for a variety of pathological conditions, but identifying metabolic checkpoints that could be exploited to manipulate the Trp metabolic network is still an unmet need. In this review, we put forward the hypothesis that pyridoxal 5'-phosphate (PLP)-dependent enzymes, which regulate multiple pathways of Trp metabolism in both the host and in microbes, might represent critical nodes and that modulating the levels of vitamin B6, from which PLP is derived, might represent a metabolic checkpoint to re-orienteer Trp flux for therapeutic purposes.

Childhood survivors of high-risk neuroblastoma show signs of immune recovery and not immunosenescence.

Neuroblastoma survivors show signs of immunosenescence early after therapy in CD8+ T cell compartment and elevated plasma TNF-α but in later follow-up immune recovery comes into play. Whether the recovery phenotype is long lasting or transient remains to be elucidated, however, late adverse effects often occur in childhood cancer survivors.

Modeling Approaches Reveal New Regulatory Networks in Aspergillus fumigatus Metabolism.

Systems biology approaches are extensively used to model and reverse-engineer gene regulatory networks from experimental data. Indoleamine 2,3-dioxygenases (IDOs)-belonging in the heme dioxygenase family-degrade l-tryptophan to kynurenines. These enzymes are also responsible for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). As such, they are expressed by a variety of species, including fungi. Interestingly, Aspergillus may degrade l-tryptophan not only via IDO but also via alternative pathways. Deciphering the molecular interactions regulating tryptophan metabolism is particularly critical for novel drug target discovery designed to control pathogen determinants in invasive infections. Using continuous time Bayesian networks over a time-course gene expression dataset, we inferred the global regulatory network controlling l-tryptophan metabolism. The method unravels a possible novel approach to target fungal virulence factors during infection. Furthermore, this study represents the first application of continuous-time Bayesian networks as a gene network reconstruction method in Aspergillus metabolism. The experiment showed that the applied computational approach may improve the understanding of metabolic networks over traditional pathways.

Microbiome-mediated regulation of anti-fungal immunity.

Anti-fungal immunity is characterized by the continuous interplay between immune activation and immune regulation processes. These processes have now been clearly shown not only in animal pre-clinical models but also in humans. To create and maintain this immune homeostasis, reciprocal interactions among the host immune system, fungal pathogens, and the microbiome are crucial. Notably, the microbiome exerts multiple direct and indirect antifungal effects that are particularly aimed at minimizing host tissue damage. Thus, in this microbiome era, the architecture of 3D culture system or 'tissue organoids' might finally represent a simple but effective in vitro 'holobiont' to unravel the diverse interactions and adaptations that evolve to overcome fungal infections.

Comparison of two human organoid models of lung and intestinal inflammation reveals Toll-like receptor signalling activation and monocyte recruitment.

OBJECTIVES: The activation of immune responses in mucosal tissues is a key factor for the development and sustainment of several pathologies including infectious diseases and autoimmune diseases. However, translational research and personalised medicine struggle to advance because of the lack of suitable preclinical models that successfully mimic the complexity of human tissues without relying on in vivo mouse models. Here, we propose two in vitro human 3D tissue models, deprived of any resident leucocytes, to model mucosal tissue inflammatory processes. METHODS: We developed human 3D lung and intestinal organoids differentiated from induced pluripotent stem cells to model mucosal tissues. We then compared their response to a panel of microbial ligands and investigated their ability to attract and host human primary monocytes. RESULTS: Mature lung and intestinal organoids comprised epithelial (EpCAM+) and mesenchymal (CD73+) cells which responded to Toll-like receptor stimulation by releasing pro-inflammatory cytokines and expressing tissue inflammatory markers including MMP9, COX2 and CRP. When added to the organoid culture, primary human monocytes migrated towards the organoids and began to differentiate to an 'intermediate-like' phenotype characterised by increased levels of CD14 and CD16. CONCLUSION: We show that human mucosal organoids exhibit proper immune functions and successfully mimic an immunocompetent tissue microenvironment able to host patient-derived immune cells. Our experimental set-up provides a novel tool to tackle the complexity of immune responses in mucosal tissues which can be tailored to different human pathologies.