CPX-351 exploits the gut microbiota to promote mucosal barrier function, colonization resistance, and immune homeostasis.

ABSTRACT: CPX-351, a liposomal combination of cytarabine plus daunorubicin, has been approved for the treatment of adults with newly diagnosed, therapy-related acute myeloid leukemia (AML) or AML with myelodysplasia-related changes, because it improves survival and outcome of patients who received hematopoietic stem cell transplant compared with the continuous infusion of cytarabine plus daunorubicin (referred to as "7 + 3" combination). Because gut dysbiosis occurring in patients with AML during induction chemotherapy heavily affects the subsequent phases of therapy, we have assessed whether the superior activity of CPX-351 vs "7 + 3" combination in the real-life setting implicates an action on and by the intestinal microbiota. To this purpose, we have evaluated the impact of CPX-351 and "7 + 3" combination on mucosal barrier function, gut microbial composition and function, and antifungal colonization resistance in preclinical models of intestinal damage in vitro and in vivo and fecal microbiota transplantation. We found that CPX-351, at variance with "7 + 3" combination, protected from gut dysbiosis, mucosal damage, and gut morbidity while increasing antifungal resistance. Mechanistically, the protective effect of CPX-351 occurred through pathways involving both the host and the intestinal microbiota, namely via the activation of the aryl hydrocarbon receptor-interleukin-22 (IL-22)-IL-10 host pathway and the production of immunomodulatory metabolites by anaerobes. This study reveals how the gut microbiota may contribute to the good safety profile, with a low infection-related mortality, of CPX-351 and highlights how a better understanding of the host-microbiota dialogue may contribute to pave the way for precision medicine in AML.

BALB/c and C57BL/6 Mice Differ in Polyreactive IgA Abundance, which Impacts the Generation of Antigen-Specific IgA and Microbiota Diversity.

The interrelationship between IgAs and microbiota diversity is still unclear. Here we show that BALB/c mice had higher abundance and diversity of IgAs than C57BL/6 mice and that this correlated with increased microbiota diversity. We show that polyreactive IgAs mediated the entrance of non-invasive bacteria to Peyer's patches, independently of CX3CR1(+) phagocytes. This allowed the induction of bacteria-specific IgA and the establishment of a positive feedback loop of IgA production. Cohousing of mice or fecal transplantation had little or no influence on IgA production and had only partial impact on microbiota composition. Germ-free BALB/c, but not C57BL/6, mice already had polyreactive IgAs that influenced microbiota diversity and selection after colonization. Together, these data suggest that genetic predisposition to produce polyreactive IgAs has a strong impact on the generation of antigen-specific IgAs and the selection and maintenance of microbiota diversity.

Aryl Hydrocarbon Receptor Agonism Antagonizes the Hypoxia-driven Inflammation in Cystic Fibrosis.

Hypoxia contributes to the exaggerated yet ineffective airway inflammation that fails to oppose infections in cystic fibrosis (CF). However, the potential for impairment of essential immune functions by HIF-1α (hypoxia-inducible factor 1α) inhibition demands a better comprehension of downstream hypoxia-dependent pathways that are amenable for manipulation. We assessed here whether hypoxia may interfere with the activity of AhR (aryl hydrocarbon receptor), a versatile environmental sensor highly expressed in the lungs, where it plays a homeostatic role. We used murine models of Aspergillus fumigatus infection in vivo and human cells in vitro to define the functional role of AhR in CF, evaluate the impact of hypoxia on AhR expression and activity, and assess whether AhR agonism may antagonize hypoxia-driven inflammation. We demonstrated that there is an important interferential cross-talk between the AhR and HIF-1α signaling pathways in murine and human CF, in that HIF-1α induction squelched the normal AhR response through an impaired formation of the AhR:ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF-1ß heterodimer. However, functional studies and analysis of the AhR genetic variability in patients with CF proved that AhR agonism could prevent hypoxia-driven inflammation, restore immune homeostasis, and improve lung function. This study emphasizes the contribution of environmental factors, such as infections, in CF disease progression and suggests the exploitation of hypoxia:xenobiotic receptor cross-talk for antiinflammatory therapy in CF.

A pathogenic role for cystic fibrosis transmembrane conductance regulator in celiac disease.

Intestinal handling of dietary proteins usually prevents local inflammatory and immune responses and promotes oral tolerance. However, in ~ 1% of the world population, gluten proteins from wheat and related cereals trigger an HLA DQ2/8-restricted TH1 immune and antibody response leading to celiac disease. Prior epithelial stress and innate immune activation are essential for breaking oral tolerance to the gluten component gliadin. How gliadin subverts host intestinal mucosal defenses remains elusive. Here, we show that the α-gliadin-derived LGQQQPFPPQQPY peptide (P31-43) inhibits the function of cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel pivotal for epithelial adaptation to cell-autonomous or environmental stress. P31-43 binds to, and reduces ATPase activity of, the nucleotide-binding domain-1 (NBD1) of CFTR, thus impairing CFTR function. This generates epithelial stress, tissue transglutaminase and inflammasome activation, NF-κB nuclear translocation and IL-15 production, that all can be prevented by potentiators of CFTR channel gating. The CFTR potentiator VX-770 attenuates gliadin-induced inflammation and promotes a tolerogenic response in gluten-sensitive mice and cells from celiac patients. Our results unveil a primordial role for CFTR as a central hub orchestrating gliadin activities and identify a novel therapeutic option for celiac disease.

Host-microbe tryptophan partitioning in cardiovascular diseases.

The functional interdependencies between the molecular components of a biological process demand for a network medicine platform that integrates systems biology and network science, to explore the interactions among biological components in health and disease. Access to large-scale omics datasets (genomics, transcriptomics, proteomics, metabolomics, metagenomics, phenomics, etc.) has significantly advanced our opportunity along this direction. Studies utilizing these techniques have begun to provide us with a deeper understanding of how the interaction between the intestinal microbes and their host affects the cardiovascular system in health and disease. Within the framework of a multiomics network approach, we highlight here how tryptophan metabolism may orchestrate the host-microbes interaction in cardiovascular diseases and the implications for precision medicine and therapeutics, including nutritional interventions.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike Protein S1 Induces Methylglyoxal-Derived Hydroimidazolone/Receptor for Advanced Glycation End Products (MG-H1/RAGE) Activation to Promote Inflammation in Human Bronchial BEAS-2B Cells.

The pathogenesis of coronavirus disease 2019 (COVID-19) is associated with a hyperinflammatory response. The mechanisms of SARS-CoV-2-induced inflammation are scantly known. Methylglyoxal (MG) is a glycolysis-derived byproduct endowed with a potent glycating action, leading to the formation of advanced glycation end products (AGEs), the main one being MG-H1. MG-H1 exerts strong pro-inflammatory effects, frequently mediated by the receptor for AGEs (RAGE). Here, we investigated the involvement of the MG-H1/RAGE axis as a potential novel mechanism in SARS-CoV-2-induced inflammation by resorting to human bronchial BEAS-2B and alveolar A549 epithelial cells, expressing different levels of the ACE2 receptor (R), exposed to SARS-CoV-2 spike protein 1 (S1). Interestingly, we found in BEAS-2B cells that do not express ACE2-R that S1 exerted a pro-inflammatory action through a novel MG-H1/RAGE-based pathway. MG-H1 levels, RAGE and IL-1ß expression levels in nasopharyngeal swabs from SARS-CoV-2-positive and -negative individuals, as well as glyoxalase 1 expression, the major scavenging enzyme of MG, seem to support the results obtained in vitro. Altogether, our findings reveal a novel mechanism involved in the inflammation triggered by S1, paving the way for the study of the MG-H1/RAGE inflammatory axis in SARS-CoV-2 infection as a potential therapeutic target to mitigate COVID-19-associated pathogenic inflammation.

From the nose to the lungs: the intricate journey of airborne pathogens amid commensal bacteria.

The recent COVID-19 pandemic has dramatically brought the pitfalls of airborne pathogens to the attention of the scientific community. Not only viruses but also bacteria and fungi may exploit air transmission to colonize and infect potential hosts and be the cause of significant morbidity and mortality in susceptible populations. The efforts to decipher the mechanisms of pathogenicity of airborne microbes have brought to light the delicate equilibrium that governs the homeostasis of mucosal membranes. The microorganisms already thriving in the permissive environment of the respiratory tract represent a critical component of this equilibrium and a potent barrier to infection by means of direct competition with airborne pathogens or indirectly via modulation of the immune response. Moving down the respiratory tract, physicochemical and biological constraints promote site-specific expansion of microbes that engage in cross talk with the local immune system to maintain homeostasis and promote protection. In this review, we critically assess the site-specific microbial communities that an airborne pathogen encounters in its hypothetical travel along the respiratory tract and discuss the changes in the composition and function of the microbiome in airborne diseases by taking fungal and SARS-CoV-2 infections as examples. Finally, we discuss how technological and bioinformatics advancements may turn microbiome analysis into a valuable tool in the hands of clinicians to predict the risk of disease onset, the clinical course, and the response to treatment of individual patients in the direction of personalized medicine implementation.

A High-Risk Profile for Invasive Fungal Infections Is Associated with Altered Nasal Microbiota and Niche Determinants.

It is becoming increasingly clear that the communities of microorganisms that populate the surfaces exposed to the external environment, termed microbiota, are key players in the regulation of pathogen-host cross talk affecting the onset as well as the outcome of infectious diseases. We have performed a multicenter, prospective, observational study in which nasal and oropharyngeal swabs were collected for microbiota predicting the risk of invasive fungal infections (IFIs) in patients with hematological malignancies. Here, we demonstrate that the nasal and oropharyngeal microbiota are different, although similar characteristics differentiate high-risk from low-risk samples at both sites. Indeed, similar to previously published results on the oropharyngeal microbiota, high-risk samples in the nose were characterized by low diversity, a loss of beneficial bacteria, and an expansion of potentially pathogenic taxa, in the presence of reduced levels of tryptophan (Trp). At variance with oropharyngeal samples, however, low Trp levels were associated with defective host-derived kynurenine production, suggesting reduced tolerance mechanisms at the nasal mucosal surface. This was accompanied by reduced levels of the chemokine interleukin-8 (IL-8), likely associated with a reduced recruitment of neutrophils and impaired fungal clearance. Thus, the nasal and pharyngeal microbiomes of hematological patients provide complementary information that could improve predictive tools for the risk of IFI in hematological patients.

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.

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.

The contribution of mast cells to bacterial and fungal infection immunity.

Mast cells are hematopoietic progenitor-derived, granule-containing immune cells that are widely distributed in tissues that interact with the external environment, such as the skin and mucosal tissues. It is well-known that mast cells are significantly involved in IgE-mediated allergic reactions, but because of their location, it has also been long hypothesized that mast cells can act as sentinel cells that sense pathogens and initiate protective immune responses. Using mast cell or mast cell protease-deficient murine models, recent studies by our groups and others indicate that mast cells have pleiotropic regulatory roles in immunological responses against pathogens. In this review, we discuss studies that demonstrate that mast cells can either promote host resistance to infections caused by bacteria and fungi or contribute to dysregulated immune responses that can increase host morbidity and mortality. Overall, these studies indicate that mast cells can influence innate immune responses against bacterial and fungal infections via multiple mechanisms. Importantly, the contribution of mast cells to infection outcomes depends in part on the infection model, including the genetic approach used to assess the influence of mast cells on host immunity, hence highlighting the complexity of mast cell biology in the context of innate immune responses.

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.

The Circadian Protein PER1 Modulates the Cellular Response to Anticancer Treatments.

The circadian clock driven by the daily light-dark and temperature cycles of the environment regulates fundamental physiological processes and perturbations of these sophisticated mechanisms may result in pathological conditions, including cancer. While experimental evidence is building up to unravel the link between circadian rhythms and tumorigenesis, it is becoming increasingly apparent that the response to antitumor agents is similarly dependent on the circadian clock, given the dependence of each drug on the circadian regulation of cell cycle, DNA repair and apoptosis. However, the molecular mechanisms that link the circadian machinery to the action of anticancer treatments is still poorly understood, thus limiting the application of circadian rhythms-driven pharmacological therapy, or chronotherapy, in the clinical practice. Herein, we demonstrate the circadian protein period 1 (PER1) and the tumor suppressor p53 negatively cross-regulate each other's expression and activity to modulate the sensitivity of cancer cells to anticancer treatments. Specifically, PER1 physically interacts with p53 to reduce its stability and impair its transcriptional activity, while p53 represses the transcription of PER1. Functionally, we could show that PER1 reduced the sensitivity of cancer cells to drug-induced apoptosis, both in vitro and in vivo in NOD scid gamma (NSG) mice xenotransplanted with a lung cancer cell line. Therefore, our results emphasize the importance of understanding the relationship between the circadian clock and tumor regulatory proteins as the basis for the future development of cancer chronotherapy.

A Shifted Composition of the Lung Microbiota Conditions the Antifungal Response of Immunodeficient Mice.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1-/- mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

Anakinra Activates Superoxide Dismutase 2 to Mitigate Inflammasome Activity.

Inflammasomes are powerful cytosolic sensors of environmental stressors and are critical for triggering interleukin-1 (IL-1)-mediated inflammatory responses. However, dysregulation of inflammasome activation may lead to pathological conditions, and the identification of negative regulators for therapeutic purposes is increasingly being recognized. Anakinra, the recombinant form of the IL-1 receptor antagonist, proved effective by preventing the binding of IL-1 to its receptor, IL-1R1, thus restoring autophagy and dampening NLR family pyrin domain containing 3 (NLRP3) activity. As the generation of mitochondrial reactive oxidative species (ROS) is a critical upstream event in the activation of NLRP3, we investigated whether anakinra would regulate mitochondrial ROS production. By profiling the activation of transcription factors induced in murine alveolar macrophages, we found a mitochondrial antioxidative pathway induced by anakinra involving the manganese-dependent superoxide dismutase (MnSOD) or SOD2. Molecularly, anakinra promotes the binding of SOD2 with the deubiquitinase Ubiquitin Specific Peptidase 36 (USP36) and Constitutive photomorphogenesis 9 (COP9) signalosome, thus increasing SOD2 protein longevity. Functionally, anakinra and SOD2 protects mice from pulmonary oxidative inflammation and infection. On a preclinical level, anakinra upregulates SOD2 in murine models of chronic granulomatous disease (CGD) and cystic fibrosis (CF). These data suggest that protection from mitochondrial oxidative stress may represent an additional mechanism underlying the clinical benefit of anakinra and identifies SOD2 as a potential therapeutic target.

Intravenous immunoglobulin protects from experimental allergic bronchopulmonary aspergillosis via a sialylation-dependent mechanism.

Intravenous immunoglobuin (IVIG) exerts protective effects in experimental allergic bronchopulmonary aspergillosis (ABPA) via a sialylation-dependent mechanism. The protection was associated with reduced recruitment of eosinophils, diminished goblet cell hyperplasia, suppressed Th2 and Th17 responses and reciprocally enhanced regulatory T cells and IL-10, and decreased IgE levels in the circulation.

Aryl hydrocarbon receptor control of a disease tolerance defence pathway.

Disease tolerance is the ability of the host to reduce the effect of infection on host fitness. Analysis of disease tolerance pathways could provide new approaches for treating infections and other inflammatory diseases. Typically, an initial exposure to bacterial lipopolysaccharide (LPS) induces a state of refractoriness to further LPS challenge (endotoxin tolerance). We found that a first exposure of mice to LPS activated the ligand-operated transcription factor aryl hydrocarbon receptor (AhR) and the hepatic enzyme tryptophan 2,3-dioxygenase, which provided an activating ligand to the former, to downregulate early inflammatory gene expression. However, on LPS rechallenge, AhR engaged in long-term regulation of systemic inflammation only in the presence of indoleamine 2,3-dioxygenase 1 (IDO1). AhR-complex-associated Src kinase activity promoted IDO1 phosphorylation and signalling ability. The resulting endotoxin-tolerant state was found to protect mice against immunopathology in Gram-negative and Gram-positive infections, pointing to a role for AhR in contributing to host fitness.

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.

Epigenetic Mechanisms of Inflammasome Regulation.

The innate immune system represents the host's first-line defense against pathogens, dead cells or environmental factors. One of the most important inflammatory pathways is represented by the activation of the NOD-like receptor (NLR) protein family. Some NLRs induce the assembly of large caspase-1-activating complexes called inflammasomes. Different types of inflammasomes have been identified that can respond to distinct bacterial, viral or fungal infections; sterile cell damage or other stressors, such as metabolic imbalances. Epigenetic regulation has been recently suggested to provide a complementary mechanism to control inflammasome activity. This regulation can be exerted through at least three main mechanisms, including CpG DNA methylation, histones post-translational modifications and noncoding RNA expression. The repression or promotion of expression of different inflammasomes (NLRP1, NLRP2, NLRP3, NLRP4, NLRP6, NLRP7, NLRP12 and AIM2) through epigenetic mechanisms determines the development of pathologies with variable severity. For example, our team recently explored the role of microRNAs (miRNAs) targeting and modulating the components of the inflammasome as potential biomarkers in bladder cancer and during therapy. This suggests that the epigenetic control of inflammasome-related genes could represent a potential target for further investigations of molecular mechanisms regulating inflammatory pathways.

Invasive mould infections in solid organ transplant patients: modifiers and indicators of disease and treatment response.

PURPOSE: Invasive mould infections, in particular invasive aspergillosis (IA), are comparatively frequent complications of immunosuppression in patients undergoing solid organ transplantation (SOT). Guidelines provide recommendations as to the procedures to be carried out to diagnose and treat IA, but only limited advice for SOT recipients. METHODS: Literature review and expert consensus summarising the existing evidence related to prophylaxis, diagnosis, treatment and assessment of response to IA and infections by Mucorales in SOT patients RESULTS: Response to therapy should be assessed early and at regular intervals. No indications of improvement should lead to a prompt change of the antifungal treatment, to account for possible infections by Mucorales or other moulds such as Scedosporium. Imaging techniques, especially CT scan and possibly angiography carried out at regular intervals during early and long-term follow-up and coupled with a careful clinical diagnostic workout, should be evaluated as diagnostic tools and outcome predictors, and standardised to improve therapy monitoring. The role of biomarkers such as the galactomannan test and PCR, as well as selected inflammation parameters, has not yet been definitively assessed in the SOT population and needs to be studied further. The therapeutic workup should consider a reduction of immunosuppressive therapy. CONCLUSIONS: The role of immunosuppression and immune tolerance mechanisms in the response to invasive fungal infection treatment is an important factor in the SOT population and should not be underestimated. The choice of the antifungal should consider not only their toxicity but also their effects on the immune system, two features that are intertwined.