Microbial metabolism of L-tyrosine protects against allergic airway inflammation.

The constituents of the gut microbiome are determined by the local habitat, which itself is shaped by immunological pressures, such as mucosal IgA. Using a mouse model of restricted antibody repertoire, we identified a role for antibody-microbe interactions in shaping a community of bacteria with an enhanced capacity to metabolize L-tyrosine. This model led to increased concentrations of p-cresol sulfate (PCS), which protected the host against allergic airway inflammation. PCS selectively reduced CCL20 production by airway epithelial cells due to an uncoupling of epidermal growth factor receptor (EGFR) and Toll-like receptor 4 (TLR4) signaling. Together, these data reveal a gut microbe-derived metabolite pathway that acts distally on the airway epithelium to reduce allergic airway responses, such as those underpinning asthma.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

Dietary Fiber Confers Protection against Flu by Shaping Ly6c- Patrolling Monocyte Hematopoiesis and CD8+ T Cell Metabolism.

Dietary fiber protects against chronic inflammatory diseases by dampening immune responses through short-chain fatty acids (SCFAs). Here we examined the effect of dietary fiber in viral infection, where the anti-inflammatory properties of SCFAs in principle could prevent protective immunity. Instead, we found that fermentable dietary fiber increased survival of influenza-infected mice through two complementary mechanisms. High-fiber diet (HFD)-fed mice exhibited altered bone marrow hematopoiesis, characterized by enhanced generation of Ly6c- patrolling monocytes, which led to increased numbers of alternatively activated macrophages with a limited capacity to produce the chemokine CXCL1 in the airways. Blunted CXCL1 production reduced neutrophil recruitment to the airways, thus limiting tissue immunopathology during infection. In parallel, diet-derived SCFAs boosted CD8+ T cell effector function by enhancing cellular metabolism. Hence, dietary fermentable fiber and SCFAs set an immune equilibrium, balancing innate and adaptive immunity so as to promote the resolution of influenza infection while preventing immune-associated pathology.

The skin microbiome in the first year of life and its association with atopic dermatitis.

BACKGROUND: Early-life microbial colonization of the skin may modulate the immune system and impact the development of atopic dermatitis (AD) and allergic diseases later in life. To address this question, we assessed the association between the skin microbiome and AD, skin barrier integrity and allergic diseases in the first year of life. We further explored the evolution of the skin microbiome with age and its possible determinants, including delivery mode. METHODS: Skin microbiome was sampled from the lateral upper arm on the first day of life, and at 3, 6, and 12 months of age. Bacterial communities were assessed by 16S rRNA gene amplicon sequencing in 346 infants from the PreventADALL population-based birth cohort study, representing 970 samples. Clinical investigations included skin examination and skin barrier function measured as trans-epidermal water loss (TEWL) at the site and time of microbiome sampling at 3, 6, and 12 months. Parental background information was recorded in electronic questionnaires, and delivery mode (including vaginal delivery (VD), VD in water, elective caesarean section (CS) and emergency CS) was obtained from maternal hospital charts. RESULTS: Strong temporal variations in skin bacterial community composition were found in the first year of life, with distinct patterns associated with different ages. Confirming our hypothesis, skin bacterial community composition in the first year of life was associated with skin barrier integrity and later onsets of AD. Delivery mode had a strong impact on the microbiome composition at birth, with each mode leading to distinct patterns of colonization. Other possible determinants of the skin microbiome were identified, including environmental and parental factors as well as breastfeeding. CONCLUSION: Skin microbiome composition during infancy is defined by age, transiently influenced by delivery mode as well as environmental, parental factors and breastfeeding. The microbiome is also associated with skin barrier integrity and the onset of AD.

Multi-omics profiling predicts allograft function after lung transplantation.

RATIONALE: Lung transplantation is the ultimate treatment option for patients with end-stage respiratory diseases but bears the highest mortality rate among all solid organ transplantations due to chronic lung allograft dysfunction (CLAD). The mechanisms leading to CLAD remain elusive due to an insufficient understanding of the complex post-transplant adaptation processes. OBJECTIVES: To better understand these lung adaptation processes after transplantation and to investigate their association with future changes in allograft function. METHODS: We performed an exploratory cohort study of bronchoalveolar lavage samples from 78 lung recipients and donors. We analysed the alveolar microbiome using 16S rRNA sequencing, the cellular composition using flow cytometry, as well as metabolome and lipidome profiling. MEASUREMENTS AND MAIN RESULTS: We established distinct temporal dynamics for each of the analysed data sets. Comparing matched donor and recipient samples, we revealed that recipient-specific as well as environmental factors, rather than the donor microbiome, shape the long-term lung microbiome. We further discovered that the abundance of certain bacterial strains correlated with underlying lung diseases even after transplantation. A decline in forced expiratory volume during the first second (FEV1) is a major characteristic of lung allograft dysfunction in transplant recipients. By using a machine learning approach, we could accurately predict future changes in FEV1 from our multi-omics data, whereby microbial profiles showed a particularly high predictive power. CONCLUSION: Bronchoalveolar microbiome, cellular composition, metabolome and lipidome show specific temporal dynamics after lung transplantation. The lung microbiome can predict future changes in lung function with high precision.

Microbiome-induced antigen-presenting cell recruitment coordinates skin and lung allergic inflammation.

BACKGROUND: Allergic skin inflammation often presents in early childhood; however, little is known about the events leading to its initiation and whether it is transient or long-term in nature. OBJECTIVE: We sought to determine the immunologic rules that govern skin inflammation in early life. METHODS: Neonatal and adult mice were epicutaneously sensitized with allergen followed by airway allergen challenge. Epicutaneous application of labeled allergen allowed for determination of antigen uptake and processing by antigen-presenting cells. RNAseq and microbiome analysis was performed on skin from neonatal and adult specific pathogen-free and germ-free mice. RESULTS: A mixed TH2/TH17 inflammatory response in the skin and the lungs of adult mice was observed following sensitization and challenge. Comparatively, neonatal mice did not develop overt skin inflammation, but exhibited systemic release of IL-17a and a TH2-dominated lung response. Mechanical skin barrier disruption was not sufficient to drive allergic skin inflammation, although it did promote systemic immune priming. Skin of neonatal mice and adult germ-free mice was seeded with low numbers of antigen-presenting cells and impaired chemokine and alarmin production. Enhanced chemokine and alarmin production, and seeding of the skin with antigen-presenting cells capable of instructing recruited cells to elicit their effector function, was, at least in part, dependent on formation of the microbiome, and consequently contributed to the development of overt skin disease. CONCLUSIONS: These data shed light on the principles that underlie allergic inflammation in different tissues and highlight a window of opportunity that might exist for early-life prevention of allergic diseases.

Airway microbiota signals anabolic and catabolic remodeling in the transplanted lung.

BACKGROUND: Homeostatic turnover of the extracellular matrix conditions the structure and function of the healthy lung. In lung transplantation, long-term management remains limited by chronic lung allograft dysfunction, an umbrella term used for a heterogeneous entity ultimately associated with pathological airway and/or parenchyma remodeling. OBJECTIVE: This study assessed whether the local cross-talk between the pulmonary microbiota and host cells is a key determinant in the control of lower airway remodeling posttransplantation. METHODS: Microbiota DNA and host total RNA were isolated from 189 bronchoalveolar lavages obtained from 116 patients post lung transplantation. Expression of a set of 11 genes encoding either matrix components or factors involved in matrix synthesis or degradation (anabolic and catabolic remodeling, respectively) was quantified by real-time quantitative PCR. Microbiota composition was characterized using 16S ribosomal RNA gene sequencing and culture. RESULTS: We identified 4 host gene expression profiles, among which catabolic remodeling, associated with high expression of metallopeptidase-7, -9, and -12, diverged from anabolic remodeling linked to maximal thrombospondin and platelet-derived growth factor D expression. While catabolic remodeling aligned with a microbiota dominated by proinflammatory bacteria (eg, Staphylococcus, Pseudomonas, and Corynebacterium), anabolic remodeling was linked to typical members of the healthy steady state (eg, Prevotella, Streptococcus, and Veillonella). Mechanistic assays provided direct evidence that these bacteria can impact host macrophage-fibroblast activation and matrix deposition. CONCLUSIONS: Host-microbes interplay potentially determines remodeling activities in the transplanted lung, highlighting new therapeutic opportunities to ultimately improve long-term lung transplant outcome.

Microbiota Promotes Chronic Pulmonary Inflammation by Enhancing IL-17A and Autoantibodies.

RATIONALE: Changes in the pulmonary microbiota are associated with progressive respiratory diseases including chronic obstructive pulmonary disease (COPD). Whether there is a causal relationship between these changes and disease progression remains unknown. OBJECTIVES: To investigate the link between an altered microbiota and disease, we used a murine model of chronic lung inflammation that is characterized by key pathological features found in COPD and compared responses in specific pathogen-free (SPF) mice and mice depleted of microbiota by antibiotic treatment or devoid of a microbiota (axenic). METHODS: Mice were challenged with LPS/elastase intranasally over 4 weeks, resulting in a chronically inflamed and damaged lung. The ensuing cellular infiltration, histological damage, and decline in lung function were quantified. MEASUREMENTS AND MAIN RESULTS: Similar to human disease, the composition of the pulmonary microbiota was altered in diseased animals. We found that the microbiota richness and diversity were decreased in LPS/elastase-treated mice, with an increased representation of the genera Pseudomonas and Lactobacillus and a reduction in Prevotella. Moreover, the microbiota was implicated in disease development as mice depleted, or devoid, of microbiota exhibited an improvement in lung function, reduced inflammation, and lymphoid neogenesis. The absence of microbial cues markedly decreased the production of IL-17A, whereas intranasal transfer of fluid enriched with the pulmonary microbiota isolated from diseased mice enhanced IL-17A production in the lungs of antibiotic-treated or axenic recipients. Finally, in mice harboring a microbiota, neutralizing IL-17A dampened inflammation and restored lung function. CONCLUSIONS: Collectively, our data indicate that host-microbial cross-talk promotes inflammation and could underlie the chronicity of inflammatory lung diseases.

Airway Microbiota Determines Innate Cell Inflammatory or Tissue Remodeling Profiles in Lung Transplantation.

RATIONALE: In lung transplant recipients, long-term graft survival relies on the control of inflammation and tissue remodeling to maintain graft functionality and avoid chronic lung allograft dysfunction. Although advances in clinical practice have improved transplant success, the mechanisms by which the balance between inflammation and remodeling is maintained are largely unknown. OBJECTIVES: To assess whether host-microbe interactions in the transplanted lung determine the immunologic tone of the airways, and consequently could impact graft survival. METHODS: Microbiota DNA and host total RNA were isolated from 203 bronchoalveolar lavages obtained from 112 patients post-lung transplantation. Microbiota composition was determined using 16S ribosomal RNA analysis, and expression of a set of genes involved in prototypic macrophage functions was quantified using real-time quantitative polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: We show that the characteristics of the pulmonary microbiota aligned with distinct innate cell gene expression profiles. Although a nonpolarized activation was associated with bacterial communities consisting of a balance between proinflammatory (e.g., Staphylococcus and Pseudomonas) and low stimulatory (e.g., Prevotella and Streptococcus) bacteria, "inflammatory" and "remodeling" profiles were linked to bacterial dysbiosis. Mechanistic assays provided direct evidence that bacterial dysbiosis could lead to inflammatory or remodeling profiles in macrophages, whereas a balanced microbial community maintained homeostasis. CONCLUSIONS: The crosstalk between bacterial communities and innate immune cells potentially determines the function of the transplanted lung offering novel pathways for intervention strategies.

Multi-omics integration reveals a nonlinear signature that precedes progression of lung fibrosis.

Objectives: Idiopathic pulmonary fibrosis (IPF) is a devastating progressive interstitial lung disease with poor outcomes. While decades of research have shed light on pathophysiological mechanisms associated with the disease, our understanding of the early molecular events driving IPF and its progression is limited. With this study, we aimed to model the leading edge of fibrosis using a data-driven approach. Methods: Multiple omics modalities (transcriptomics, metabolomics and lipidomics) of healthy and IPF lung explants representing different stages of fibrosis were combined using an unbiased approach. Multi-Omics Factor Analysis of datasets revealed latent factors specifically linked with established fibrotic disease (Factor1) and disease progression (Factor2). Results: Features characterising Factor1 comprised well-established hallmarks of fibrotic disease such as defects in surfactant, epithelial-mesenchymal transition, extracellular matrix deposition, mitochondrial dysfunction and purine metabolism. Comparatively, Factor2 identified a signature revealing a nonlinear trajectory towards disease progression. Molecular features characterising Factor2 included genes related to transcriptional regulation of cell differentiation, ciliogenesis and a subset of lipids from the endocannabinoid class. Machine learning models, trained upon the top transcriptomics features of each factor, accurately predicted disease status and progression when tested on two independent datasets. Conclusion: This multi-omics integrative approach has revealed a unique signature which may represent the inflection point in disease progression, representing a promising avenue for the identification of therapeutic targets aimed at addressing the progressive nature of the disease.

Butyrate regulates neutrophil homeostasis and impairs early antimicrobial activity in the lung.

Short-chain fatty acids (SCFAs) are metabolites that are produced after microbial fermentation of dietary fiber and impact cell metabolism and anti-inflammatory pathways both locally in the gut and systemically. In preclinical models, administration of SCFAs, such as butyrate, ameliorates a range of inflammatory disease models including allergic airway inflammation, atopic dermatitis, and influenza infection. Here we report the effect of butyrate on a bacteria-induced acute neutrophil-driven immune response in the airways. Butyrate impacted discrete aspects of hematopoiesis in the bone marrow resulting in the accumulation of immature neutrophils. During Pseudomonas aeruginosa infection, butyrate treatment led to the enhanced mobilization of neutrophils to the lungs as a result of increased CXCL2 expression by lung macrophages. Despite this increase in granulocyte numbers and their enhanced phagocytic capacity, neutrophils failed to control early bacterial growth. Butyrate reduced the expression of nicotinamide adenine dinucleotide phosphate, oxidase complex components required for reactive oxygen species production, and reduced secondary granule enzymes, culminating in impaired bactericidal activity. These data reveal that SCFAs tune neutrophil maturation and effector function in the bone marrow under homeostatic conditions, potentially to mitigate against excessive granulocyte-driven immunopathology, but their consequently restricted bactericidal capacity impairs early control of Pseudomonas infection.

Early life inter-kingdom interactions shape the immunological environment of the airways.

BACKGROUND: There is increasing evidence that the airway microbiome plays a key role in the establishment of respiratory health by interacting with the developing immune system early in life. While it has become clear that bacteria are involved in this process, there is a knowledge gap concerning the role of fungi. Moreover, the inter-kingdom interactions that influence immune development remain unknown. In this prospective exploratory human study, we aimed to determine early post-natal microbial and immunological features of the upper airways in 121 healthy newborns. RESULTS: We found that the oropharynx and nasal cavity represent distinct ecological niches for bacteria and fungi. Breastfeeding correlated with changes in microbiota composition of oropharyngeal samples with the greatest impact upon the relative abundance of Streptococcus species and Candida. Host transcriptome profiling revealed that genes with the highest expression variation were immunological in nature. Multi-omics factor analysis of host and microbial data revealed unique co-variation patterns. CONCLUSION: These data provide evidence of a diverse multi-kingdom microbiota linked with local immunological characteristics in the first week of life that could represent distinct trajectories for future respiratory health. TRIAL REGISTRATION: NHS Health Research Authority, IRAS ID 199053. Registered 5 Oct 2016. https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/breathing-together/ Video abstract.

A prevalent and culturable microbiota links ecological balance to clinical stability of the human lung after transplantation.

There is accumulating evidence that the lower airway microbiota impacts lung health. However, the link between microbial community composition and lung homeostasis remains elusive. We combine amplicon sequencing and bacterial culturing to characterize the viable bacterial community in 234 longitudinal bronchoalveolar lavage samples from 64 lung transplant recipients and establish links to viral loads, host gene expression, lung function, and transplant health. We find that the lung microbiota post-transplant can be categorized into four distinct compositional states, 'pneumotypes'. The predominant 'balanced' pneumotype is characterized by a diverse bacterial community with moderate viral loads, and host gene expression profiles suggesting immune tolerance. The other three pneumotypes are characterized by being either microbiota-depleted, or dominated by potential pathogens, and are linked to increased immune activity, lower respiratory function, and increased risks of infection and rejection. Collectively, our findings establish a link between the lung microbial ecosystem, human lung function, and clinical stability post-transplant.

Toll-Interacting Protein Regulates Immune Cell Infiltration and Promotes Colitis-Associated Cancer.

Expression of Toll-interacting protein (Tollip), a potent TLR modulator, decreases in patients with inflammatory bowel diseases (IBD), whereas Tollip-/- mice are susceptible to colitis. Tollip expression was shown to be reduced in sporadic adenoma. In contrast, we found variable Tollip expression in patients with colitis-associated adenomas. In Tollip-/- mice challenged to develop colitis-associated cancer (CAC), tumor formation was significantly reduced owing to decreased mucosal proliferative and apoptotic indexes. This protection was associated with blunt inflammatory responses without significant changes in microbial composition. mRNA expression of Cd62l and Ccr5 homing receptors was reduced in colons of untreated Tollip-/- mice, whereas CD62L+ CD8+ T cells accumulated in the periphery. In Tollip-deficient adenomas Ctla-4 mRNA expression and tumor-infiltrating CD4+ Foxp3+ regulatory T cell (Treg) were decreased. Our data show that protection from CAC in Tollip-deficient mice is associated with defects in lymphocyte accumulation and composition in colitis-associated adenomas.

Early origins of lung disease: towards an interdisciplinary approach.

The prenatal and perinatal environments can have profound effects on the development of chronic inflammatory diseases. However, mechanistic insight into how the early-life microenvironment can impact upon development of the lung and immune system and consequent initiation and progression of respiratory diseases is still emerging. Recent studies investigating the developmental origins of lung diseases have started to delineate the effects of early-life changes in the lung, environmental exposures and immune maturation on the development of childhood and adult lung diseases. While the influencing factors have been described and studied in mostly animal models, it remains challenging to pinpoint exactly which factors and at which time point are detrimental in lung development leading to respiratory disease later in life. To advance our understanding of early origins of chronic lung disease and to allow for proper dissemination and application of this knowledge, we propose four major focus areas: 1) policy and education; 2) clinical assessment; 3) basic and translational research; and 4) infrastructure and tools, and discuss future directions for advancement. This review is a follow-up of the discussions at the European Respiratory Society Research Seminar "Early origins of lung disease: towards an interdisciplinary approach" (Lisbon, Portugal, November 2019).

Sperm Microbiota and Its Impact on Semen Parameters.

Compared to its female counterpart, the microbiota of the male genital tract has not been studied extensively. With this study, we aimed to evaluate the bacterial composition of seminal fluid and its impact on sperm parameters. We hypothesized that a dysbiotic microbiota composition may have an influence on sperm quality. Semen samples of 26 men with normal spermiogram and 68 men with at least one abnormal spermiogram parameter were included in the study. Samples were stratified based on total sperm count, spermatozoa concentration, progressive motility, total motility and spermatozoa morphology. Microbiota profiling was performed using 16S rRNA gene amplicons sequencing and total bacterial load was determined using a panbacterial quantitative PCR. Semen samples broadly clustered into three microbiota profiles: Prevotella-enriched, Lactobacillus-enriched, and polymicrobial. Prevotella-enriched samples had the highest bacterial load (p < 0.05). Network analysis identified three main co-occurrence modules, among which two contained bacteria commonly found in the vaginal flora. Genera from the same module displayed similar oxygen requirements, arguing for the presence of different ecological niches for bacteria that colonize semen through the passage. Contrary to our hypothesis, shifts in overall microbiota composition (beta-diversity) did not correlate with spermiogram parameters. Similarly, we did not find any difference in microbial richness or diversity (alpha-diversity). Differential abundance testing, however, revealed three specific genera that were significantly enriched or depleted in some of the sperm quality groups (p < 0.05). Prevotella relative abundance was increased in samples with defective sperm motility while Staphylococcus was increased in the corresponding control group. In addition, we observed an increased relative abundance of Lactobacillus in samples with normal sperm morphology. Our study indicates that overall bacterial content of sperm might not play a major role in male infertility. Although no major shifts in microbiota composition or diversity were found, the differential abundance of specific bacterial genera in the sperm suggests that a small subset of microbes might impact the spermatozoal physiology during sperm transition, more specifically motility and morphology. Further studies are required to challenge this finding and develop potential strategies to induce the formation of a healthy seminal microbiota.

Early-Life Formation of the Microbial and Immunological Environment of the Human Airways.

Crosstalk between immune cells and the microbiota in mucosal tissues can set an individual on a trajectory toward health or disease. Little is known about these early-life events in the human respiratory tract. We examined bacterial colonization and immune system maturation in the lower airways over the first year of life. The lower respiratory tract microbiota forms within the first 2 postnatal months. Within the first weeks, three microbial profiles are evident, broadly distinguished as dysbiotic or diverse, and representing different microbial virulence potentials, including proteolysis of immunoglobulin A (IgA) that is critical for mucosal defense. Delivery mode determines microbiota constituents in preterm, but not term, births. Gestational age is a key determinant of immune maturation, with airway cells progressively increasing expression of proallergic cytokine interleukin-33 and genes linked with IgA. These data reveal microbial and immunological development in human airways, and may inform early-life interventions to prevent respiratory diseases.

Microbiota Analysis Using an Illumina MiSeq Platform to Sequence 16S rRNA Genes.

The microbiota have been shown to play an important role in diverse biological processes including immunity, metabolism, and digestion. Assessing the exact composition of the microbiota has proven challenging due to the often unknown growth specificities of its members, and culture-based approaches typically fail to capture the complete diversity of microorganisms present. Next Generation Sequencing (NGS) methods provide an efficient means to gather information about cultured and uncultured members of the microbiota. This article provides a method to characterize bacterial communities in terms of species composition using high-throughput sequencing. Briefly, by extracting the entire DNA content of a microbiota sample and performing a targeted high-throughput sequencing of the 16S rRNA gene, a phylogenetic marker for prokaryotes, prediction of the composition of the entire bacterial community is made possible. © 2017 by John Wiley & Sons, Inc.

Asthma Prevention: Right Bugs, Right Time?

Asthma is an increasingly prevalent chronic respiratory disease particularly affecting children. Microbial colonization during early life has emerged as a central factor influencing asthma susceptibility. A recently published prospective study links the reduction in relative abundance of 4 bacterial genera at 3 months of age to the development of asthma.