A gut microbial signature for combination immune checkpoint blockade across cancer types.

Immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte protein 4 (CTLA-4) can induce remarkable, yet unpredictable, responses across a variety of cancers. Studies suggest that there is a relationship between a cancer patient's gut microbiota composition and clinical response to ICB; however, defining microbiome-based biomarkers that generalize across cohorts has been challenging. This may relate to previous efforts quantifying microbiota to species (or higher taxonomic rank) abundances, whereas microbial functions are often strain specific. Here, we performed deep shotgun metagenomic sequencing of baseline fecal samples from a unique, richly annotated phase 2 trial cohort of patients with diverse rare cancers treated with combination ICB (n = 106 discovery cohort). We demonstrate that strain-resolved microbial abundances improve machine learning predictions of ICB response and 12-month progression-free survival relative to models built using species-rank quantifications or comprehensive pretreatment clinical factors. Through a meta-analysis of gut metagenomes from a further six comparable studies (n = 364 validation cohort), we found cross-cancer (and cross-country) validity of strain-response signatures, but only when the training and test cohorts used concordant ICB regimens (anti-PD-1 monotherapy or combination anti-PD-1 plus anti-CTLA-4). This suggests that future development of gut microbiome diagnostics or therapeutics should be tailored according to ICB treatment regimen rather than according to cancer type.

Genomic attributes of airway commensal bacteria and mucosa.

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

Multi-level analysis of the gut-brain axis shows autism spectrum disorder-associated molecular and microbial profiles.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by heterogeneous cognitive, behavioral and communication impairments. Disruption of the gut-brain axis (GBA) has been implicated in ASD although with limited reproducibility across studies. In this study, we developed a Bayesian differential ranking algorithm to identify ASD-associated molecular and taxa profiles across 10 cross-sectional microbiome datasets and 15 other datasets, including dietary patterns, metabolomics, cytokine profiles and human brain gene expression profiles. We found a functional architecture along the GBA that correlates with heterogeneity of ASD phenotypes, and it is characterized by ASD-associated amino acid, carbohydrate and lipid profiles predominantly encoded by microbial species in the genera Prevotella, Bifidobacterium, Desulfovibrio and Bacteroides and correlates with brain gene expression changes, restrictive dietary patterns and pro-inflammatory cytokine profiles. The functional architecture revealed in age-matched and sex-matched cohorts is not present in sibling-matched cohorts. We also show a strong association between temporal changes in microbiome composition and ASD phenotypes. In summary, we propose a framework to leverage multi-omic datasets from well-defined cohorts and investigate how the GBA influences ASD.

Screening for Clostridioides difficile colonization at admission to the hospital: a multi-centre study.

OBJECTIVES: To assess the value of screening for Clostridioides difficile colonization (CDC) at hospital admission in an endemic setting. METHODS: A multi-centre study was conducted at four hospitals located across the Netherlands. Newly admitted patients were screened for CDC. The risk of development of Clostridioides difficile infection (CDI) during admission and 1-year follow-up was assessed in patients with and without colonization. C. difficile isolates from patients with colonization were compared with isolates from incident CDI cases using core genome multi-locus sequence typing to determine whether onwards transmission had occurred. RESULTS: CDC was present in 108 of 2211 admissions (4.9%), whereas colonization with a toxigenic strain (toxigenic Clostridoides difficile colonization [tCDC]) was present in 68 of 2211 admissions (3.1%). Among these 108 patients with colonization, diverse PCR ribotypes were found and no 'hypervirulent' PCR ribotype 027 (RT027) was detected (95% CI, 0-0.028). None of the patients with colonization developed CDI during admission (0/49; 95% CI, 0-0.073) or 1-year follow-up (0/38; 95% CI, 0-0.93). Core genome multi-locus sequence typing identified six clusters with genetically related isolates from patients with tCDC and CDI; however, in these clusters, only one possible transmission event from a patient with tCDC to a patient with CDI was identified based on epidemiological data. CONCLUSION: In this endemic setting with a low prevalence of 'hypervirulent' strains, screening for CDC at admission did not detect any patients with CDC who progressed to symptomatic CDI and detected only one possible transmission event from a patient with colonization to a patient with CDI. Thus, screening for CDC at admission is not useful in this setting.

Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies.

Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. Here we evaluate recent studies that characterized microbial populations in human fetuses from the perspectives of reproductive biology, microbial ecology, bioinformatics, immunology, clinical microbiology and gnotobiology, and assess possible mechanisms by which the fetus might interact with microorganisms. Our analysis indicates that the detected microbial signals are likely the result of contamination during the clinical procedures to obtain fetal samples or during DNA extraction and DNA sequencing. Furthermore, the existence of live and replicating microbial populations in healthy fetal tissues is not compatible with fundamental concepts of immunology, clinical microbiology and the derivation of germ-free mammals. These conclusions are important to our understanding of human immune development and illustrate common pitfalls in the microbial analyses of many other low-biomass environments. The pursuit of a fetal microbiome serves as a cautionary example of the challenges of sequence-based microbiome studies when biomass is low or absent, and emphasizes the need for a trans-disciplinary approach that goes beyond contamination controls by also incorporating biological, ecological and mechanistic concepts.

Intelectin-1 binds and alters the localization of the mucus barrier-modifying bacterium Akkermansia muciniphila.

Intelectin-1 (ITLN1) is a lectin secreted by intestinal epithelial cells (IECs) and upregulated in human ulcerative colitis (UC). We investigated how ITLN1 production is regulated in IECs and the biological effects of ITLN1 at the host-microbiota interface using mouse models. Our data show that ITLN1 upregulation in IECs from UC patients is a consequence of activating the unfolded protein response. Analysis of microbes coated by ITLN1 in vivo revealed a restricted subset of microorganisms, including the mucolytic bacterium Akkermansia muciniphila. Mice overexpressing intestinal ITLN1 exhibited decreased inner colonic mucus layer thickness and closer apposition of A. muciniphila to the epithelial cell surface, similar to alterations reported in UC. The changes in the inner mucus layer were microbiota and A. muciniphila dependent and associated with enhanced sensitivity to chemically induced and T cell-mediated colitis. We conclude that by determining the localization of a select group of bacteria to the mucus layer, ITLN1 modifies this critical barrier. Together, these findings may explain the impact of ITLN1 dysregulation on UC pathogenesis.

The heightened importance of the microbiome in cancer immunotherapy.

The human microbiome is recognized as a key factor in health and disease. This has been further corroborated by identifying changes in microbiome composition and function as a novel hallmark in cancer. These effects are exerted through microbiome interactions with host cells, impacting a wide variety of developmental and physiological processes. In this review, we discuss some of the latest findings on how the bacterial component of the microbiome can influence outcomes for different cancer immunotherapy modalities, highlighting identified mechanisms of action. We also address the clinical efforts to utilize this knowledge to achieve better responses to immunotherapy. A refined understanding of microbiome variations in patients and microbiome-host interactions with cancer therapies is essential to realize optimal clinical responses.

Strong pathogen competition in neonatal gut colonisation.

Opportunistic bacterial pathogen species and their strains that colonise the human gut are generally understood to compete against both each other and the commensal species colonising this ecosystem. Currently we are lacking a population-wide quantification of strain-level colonisation dynamics and the relationship of colonisation potential to prevalence in disease, and how ecological factors might be modulating these. Here, using a combination of latest high-resolution metagenomics and strain-level genomic epidemiology methods we performed a characterisation of the competition and colonisation dynamics for a longitudinal cohort of neonatal gut microbiomes. We found strong inter- and intra-species competition dynamics in the gut colonisation process, but also a number of synergistic relationships among several species belonging to genus Klebsiella, which includes the prominent human pathogen Klebsiella pneumoniae. No evidence of preferential colonisation by hospital-adapted pathogen lineages in either vaginal or caesarean section birth groups was detected. Our analysis further enabled unbiased assessment of strain-level colonisation potential of extra-intestinal pathogenic Escherichia coli (ExPEC) in comparison with their propensity to cause bloodstream infections. Our study highlights the importance of systematic surveillance of bacterial gut pathogens, not only from disease but also from carriage state, to better inform therapies and preventive medicine in the future.

The Childhood Acute Illness and Nutrition (CHAIN) network nested case-cohort study protocol: a multi-omics approach to understanding mortality among children in sub-Saharan Africa and South Asia.

Introduction: Many acutely ill children in low- and middle-income settings have a high risk of mortality both during and after hospitalisation despite guideline-based care. Understanding the biological mechanisms underpinning mortality may suggest optimal pathways to target for interventions to further reduce mortality. The Childhood Acute Illness and Nutrition (CHAIN) Network ( www.chainnnetwork.org) Nested Case-Cohort Study (CNCC) aims to investigate biological mechanisms leading to inpatient and post-discharge mortality through an integrated multi-omic approach. Methods and analysis; The CNCC comprises a subset of participants from the CHAIN cohort (1278/3101 hospitalised participants, including 350 children who died and 658 survivors, and 270/1140 well community children of similar age and household location) from nine sites in six countries across sub-Saharan Africa and South Asia. Systemic proteome, metabolome, lipidome, lipopolysaccharides, haemoglobin variants, toxins, pathogens, intestinal microbiome and biomarkers of enteropathy will be determined. Computational systems biology analysis will include machine learning and multivariate predictive modelling with stacked generalization approaches accounting for the different characteristics of each biological modality. This systems approach is anticipated to yield mechanistic insights, show interactions and behaviours of the components of biological entities, and help develop interventions to reduce mortality among acutely ill children. Ethics and dissemination. The CHAIN Network cohort and CNCC was approved by institutional review boards of all partner sites. Results will be published in open access, peer reviewed scientific journals and presented to academic and policy stakeholders. Data will be made publicly available, including uploading to recognised omics databases. Trial registration NCT03208725.

The gut microbial metabolic capacity of microbiome-humanized vs. wild type rodents reveals a likely dual role of intestinal bacteria in hepato-intestinal schistosomiasis.

Increasing evidence shows that the host gut microbiota might be involved in the immunological cascade that culminates with the formation of tissue granulomas underlying the pathophysiology of hepato-intestinal schistosomiasis. In this study, we investigated the impact of Schistosoma mansoni infection on the gut microbial composition and functional potential of both wild type and microbiome-humanized mice. In spite of substantial differences in microbiome composition at baseline, selected pathways were consistently affected by parasite infection. The gut microbiomes of infected mice of both lines displayed, amongst other features, enhanced capacity for tryptophan and butyrate production, which might be linked to the activation of mechanisms aimed to prevent excessive injuries caused by migrating parasite eggs. Complementing data from previous studies, our findings suggest that the host gut microbiome might play a dual role in the pathophysiology of schistosomiasis, where intestinal bacteria may contribute to egg-associated pathology while, in turn, protect the host from uncontrolled tissue damage.

The association between early-life gut microbiota and childhood respiratory diseases: a systematic review.

Data from animal models suggest a role of early-life gut microbiota in lung immune development, and in establishing susceptibility to respiratory infections and asthma in humans. This systematic review summarises the association between infant (ages 0-12 months) gut microbiota composition measured by genomic sequencing, and childhood (ages 0-18 years) respiratory diseases (ie, respiratory infections, wheezing, or asthma). Overall, there was evidence that low α-diversity and relative abundance of particular gut-commensal bacteria genera (Bifidobacterium, Faecalibacterium, Ruminococcus, and Roseburia) are associated with childhood respiratory diseases. However, results were inconsistent and studies had important limitations, including insufficient characterisation of bacterial taxa to species level, heterogeneous outcome definitions, residual confounding, and small sample sizes. Large longitudinal studies with stool sampling during the first month of life and shotgun metagenomic approaches to improve bacterial and fungal taxa resolution are needed. Standardising follow-up times and respiratory disease definitions and optimising causal statistical approaches might identify targets for primary prevention of childhood respiratory diseases.

Mother-infant transmission of human microbiota.

Humans are colonised by a highly adapted microbiota with coevolved functions that promote human health, development and disease resistance. Acquisition and assembly of the microbiota start at birth and recent evidence suggests that it coincides with, and informs, immune system development and regulation in the rapidly growing infant. Several large-scale studies have identified Bifidobacterium and Bacteroides species maternally transmitted to infants, many of which are capable of colonising over the longer term. Disruption of maternal transmission by caesarean section and antibiotic exposure around birth is associated with a higher incidence of pathogen colonisation and immune-related disorders in children. In this review, we discuss key maternally transmitted bacterial species, their sources and their potential role in shaping immune development. Maternal transmission of gut bacteria provides a microbial 'starter kit' for infants which promotes healthy growth and disease resistance. Optimising and nurturing this under-appreciated form of kinship should be considered as a priority.

'Know thyself' - host factors influencing cancer response to immune checkpoint inhibitors.

Immune checkpoint inhibitors (ICIs) have revolutionised oncology and are now standard-of-care for the treatment of a wide variety of solid neoplasms. However, tumour responses remain unpredictable, experienced by only a minority of ICI recipients across malignancy types. Therefore, there is an urgent need for better predictive biomarkers to identify a priori the patients most likely to benefit from these therapies. Despite considerable efforts, only three such biomarkers are FDA-approved for clinical use, and all rely on the availability of tumour tissue for immunohistochemical staining or genomic assays. There is emerging evidence that host factors - for example, genetic, metabolic, and immune factors, as well as the composition of one's gut microbiota - influence the response of a patient's cancer to ICIs. Tantalisingly, some of these factors are modifiable, paving the way for co-therapies that may enhance the therapeutic index of these treatments. Herein, we review key host factors that are of potential biomarker value for response to ICI therapy, with a particular focus on the proposed mechanisms for these influences. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identification of gut microbial species linked with disease variability in a widely used mouse model of colitis.

Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.

Strain-level characterization of broad host range mobile genetic elements transferring antibiotic resistance from the human microbiome.

Mobile genetic elements (MGEs) carrying antibiotic resistance genes (ARGs) disseminate ARGs when they mobilise into new bacterial hosts. The nature of such horizontal gene transfer (HGT) events between human gut commensals and pathogens remain poorly characterised. Here, we compare 1354 cultured commensal strains (540 species) to 45,403 pathogen strains (12 species) and find 64,188 MGE-mediated ARG transfer events between the two groups using established methods. Among the 5931 MGEs, we find 15 broad host range elements predicted to have crossed different bacterial phyla while also occurring in animal and environmental microbiomes. We experimentally demonstrate that predicted broad host range MGEs can mobilise from commensals Dorea longicatena and Hungatella hathewayi to pathogen Klebsiella oxytoca, crossing phyla simultaneously. Our work establishes the MGE-mediated ARG dissemination network between human gut commensals and pathogens and highlights broad host range MGEs as targets for future ARG dissemination management.

A purine metabolic checkpoint that prevents autoimmunity and autoinflammation.

Still's disease, the paradigm of autoinflammation-cum-autoimmunity, predisposes for a cytokine storm with excessive T lymphocyte activation upon viral infection. Loss of function of the purine nucleoside enzyme FAMIN is the sole known cause for monogenic Still's disease. Here we discovered that a FAMIN-enabled purine metabolon in dendritic cells (DCs) restrains CD4+ and CD8+ T cell priming. DCs with absent FAMIN activity prime for enhanced antigen-specific cytotoxicity, IFNγ secretion, and T cell expansion, resulting in excessive influenza A virus-specific responses. Enhanced priming is already manifest with hypomorphic FAMIN-I254V, for which ∼6% of mankind is homozygous. FAMIN controls membrane trafficking and restrains antigen presentation in an NADH/NAD+-dependent manner by balancing flux through adenine-guanine nucleotide interconversion cycles. FAMIN additionally converts hypoxanthine into inosine, which DCs release to dampen T cell activation. Compromised FAMIN consequently enhances immunosurveillance of syngeneic tumors. FAMIN is a biochemical checkpoint that protects against excessive antiviral T cell responses, autoimmunity, and autoinflammation.

The MITRE trial protocol: a study to evaluate the microbiome as a biomarker of efficacy and toxicity in cancer patients receiving immune checkpoint inhibitor therapy.

BACKGROUND: The gut microbiome is implicated as a marker of response to immune checkpoint inhibitors (ICI) based on preclinical mouse models and preliminary observations in limited patient series. Furthermore, early studies suggest faecal microbial transfer may have therapeutic potential, converting ICI non-responders into responders. So far, identification of specific responsible bacterial taxa has been inconsistent, which limits future application. The MITRE study will explore and validate a microbiome signature in a larger scale prospective study across several different cancer types. METHODS: Melanoma, renal cancer and non-small cell lung cancer patients who are planned to receive standard immune checkpoint inhibitors are being recruited to the MITRE study. Longitudinal stool samples are collected prior to treatment, then at 6 weeks, 3, 6 and 12 months during treatment, or at disease progression/recurrence (whichever is sooner), as well as after a severe (≥grade 3 CTCAE v5.0) immune-related adverse event. Additionally, whole blood, plasma, buffy coat, RNA and peripheral blood mononuclear cells (PBMCs) is collected at similar time points and will be used for exploratory analyses. Archival tumour tissue, tumour biopsies at progression/relapse, as well as any biopsies from body organs collected after a severe toxicity are collected. The primary outcome measure is the ability of the microbiome signature to predict 1 year progression-free survival (PFS) in patients with advanced disease. Secondary outcomes include microbiome correlations with toxicity and other efficacy end-points. Biosamples will be used to explore immunological and genomic correlates. A sub-study will evaluate both COVID-19 antigen and antibody associations with the microbiome. DISCUSSION: There is an urgent need to identify biomarkers that are predictive of treatment response, resistance and toxicity to immunotherapy. The data generated from this study will both help inform patient selection for these drugs and provide information that may allow therapeutic manipulation of the microbiome to improve future patient outcomes. TRIAL REGISTRATION: NCT04107168 , ClinicalTrials.gov, registered 09/27/2019. Protocol V3.2 (16/04/2021).