Toxigenic Clostridium perfringens isolated from at-risk pediatric inflammatory bowel disease patients.

BACKGROUND AND AIMS: The goal was to identify microbial drivers of IBD, by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from pediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying C. perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in 8 of 9 patients' mucosal biopsies, correlating with hemolytic activity, while not in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults (18.7-27.1%) versus healthy (5.1%). In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial, neuroblasts, and neutrophils, while impact on epithelial cells was less pronounced, suggesting C. perfringens may be damaging particularly when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed PFO toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.

Melanoma and microbiota: Current understanding and future directions.

Over the last decade, the composition of the gut microbiota has been found to correlate with the outcomes of cancer patients treated with immunotherapy. Accumulating evidence points to the various mechanisms by which intestinal bacteria act on distal tumors and how to harness this complex ecosystem to circumvent primary resistance to immune checkpoint inhibitors. Here, we review the state of the microbiota field in the context of melanoma, the recent breakthroughs in defining microbial modes of action, and how to modulate the microbiota to enhance response to cancer immunotherapy. The host-microbe interaction may be deciphered by the use of "omics" technologies, and will guide patient stratification and the development of microbiota-centered interventions. Efforts needed to advance the field and current gaps of knowledge are also discussed.

Enrichment of oral-derived bacteria in inflamed colorectal tumors and distinct associations of Fusobacterium in the mesenchymal subtype.

While the association between colorectal cancer (CRC) features and Fusobacterium has been extensively studied, less is known of other intratumoral bacteria. Here, we leverage whole transcriptomes from 807 CRC samples to dually characterize tumor gene expression and 74 intratumoral bacteria. Seventeen of these species, including 4 Fusobacterium spp., are classified as orally derived and are enriched among right-sided, microsatellite instability-high (MSI-H), and BRAF-mutant tumors. Across consensus molecular subtypes (CMSs), integration of Fusobacterium animalis (Fa) presence and tumor expression reveals that Fa has the most significant associations in mesenchymal CMS4 tumors despite a lower prevalence than in immune CMS1. Within CMS4, the prevalence of Fa is uniquely associated with collagen- and immune-related pathways. Additional Fa pangenome analysis reveals that stress response genes and the adhesion FadA are commonly expressed intratumorally. Overall, this study identifies oral-derived bacteria as enriched in inflamed tumors, and the associations of bacteria and tumor expression are context and species specific.

Microbial genetic and transcriptional contributions to oxalate degradation by the gut microbiota in health and disease.

Over-accumulation of oxalate in humans may lead to nephrolithiasis and nephrocalcinosis. Humans lack endogenous oxalate degradation pathways (ODP), but intestinal microbes can degrade oxalate using multiple ODPs and protect against its absorption. The exact oxalate-degrading taxa in the human microbiota and their ODP have not been described. We leverage multi-omics data (>3000 samples from >1000 subjects) to show that the human microbiota primarily uses the type II ODP, rather than type I. Furthermore, among the diverse ODP-encoding microbes, an oxalate autotroph, Oxalobacter formigenes, dominates this function transcriptionally. Patients with inflammatory bowel disease (IBD) frequently suffer from disrupted oxalate homeostasis and calcium oxalate nephrolithiasis. We show that the enteric oxalate level is elevated in IBD patients, with highest levels in Crohn's disease (CD) patients with both ileal and colonic involvement consistent with known nephrolithiasis risk. We show that the microbiota ODP expression is reduced in IBD patients, which may contribute to the disrupted oxalate homeostasis. The specific changes in ODP expression by several important taxa suggest that they play distinct roles in IBD-induced nephrolithiasis risk. Lastly, we colonize mice that are maintained in the gnotobiotic facility with O. formigenes, using either a laboratory isolate or an isolate we cultured from human stools, and observed a significant reduction in host fecal and urine oxalate levels, supporting our in silico prediction of the importance of the microbiome, particularly O. formigenes in host oxalate homeostasis.

Biomarker Technologies to Support Early Clinical Immuno-oncology Development: Advances and Interpretation.

Today, there is a huge effort to develop cancer immunotherapeutics capable of combating cancer cells as well as the biological environment in which they can grow, adapt, and survive. For such treatments to benefit more patients, there is a great need to dissect the complex interplays between tumor cells and the host's immune system. Monitoring mechanisms of resistance to immunotherapeutics can delineate the evolution of key players capable of driving an efficacious antitumor immune response. In doing so, simultaneous and systematic interrogation of multiple biomarkers beyond single biomarker approaches needs to be undertaken. Zooming into cell-to-cell interactions using technological advancements with unprecedented cellular resolution such as single-cell spatial transcriptomics, advanced tissue histology approaches, and new molecular immune profiling tools promises to provide a unique level of molecular granularity of the tumor environment and may support better decision-making during drug development. This review will focus on how such technological tools are applied in clinical settings, to inform the underlying tumor-immune biology of patients and offer a deeper understanding of cancer immune responsiveness to immuno-oncology treatments.

Gut microbiome stability and dynamics in healthy donors and patients with non-gastrointestinal cancers.

As microbial therapeutics are increasingly being tested in diverse patient populations, it is essential to understand the host and environmental factors influencing the microbiome. Through analysis of 1,359 gut microbiome samples from 946 healthy donors of the Milieu Intérieur cohort, we detail how microbiome composition is associated with host factors, lifestyle parameters, and disease states. Using a genome-based taxonomy, we found biological sex was the strongest driver of community composition. Additionally, bacterial populations shift across decades of life (age 20-69), with Bacteroidota species consistently increased with age while Actinobacteriota species, including Bifidobacterium, decreased. Longitudinal sampling revealed that short-term stability exceeds interindividual differences. By accounting for these factors, we defined global shifts in the microbiomes of patients with non-gastrointestinal tumors compared with healthy donors. Together, these results demonstrated that the microbiome displays predictable variations as a function of sex, age, and disease state. These variations must be considered when designing microbiome-targeted therapies or interpreting differences thought to be linked to pathophysiology or therapeutic response.

A Cutibacterium acnes antibiotic modulates human skin microbiota composition in hair follicles.

The composition of the skin microbiota varies widely among individuals when sampled at the same body site. A key question is which molecular factors determine strain-level variability within sub-ecosystems of the skin microbiota. Here, we used a genomics-guided approach to identify an antibacterial biosynthetic gene cluster in Cutibacterium acnes (formerly Propionibacterium acnes), a human skin commensal bacterium that is widely distributed across individuals and skin sites. Experimental characterization of this biosynthetic gene cluster resulted in identification of a new thiopeptide antibiotic, cutimycin. Analysis of individual human skin hair follicles revealed that cutimycin contributed to the ecology of the skin hair follicle microbiota and helped to reduce colonization of skin hair follicles by Staphylococcus species.

A comprehensive assessment of demographic, environmental, and host genetic associations with gut microbiome diversity in healthy individuals.

BACKGROUND: The gut microbiome is an important determinant of human health. Its composition has been shown to be influenced by multiple environmental factors and likely by host genetic variation. In the framework of the Milieu Intérieur Consortium, a total of 1000 healthy individuals of western European ancestry, with a 1:1 sex ratio and evenly stratified across five decades of life (age 20-69), were recruited. We generated 16S ribosomal RNA profiles from stool samples for 858 participants. We investigated genetic and non-genetic factors that contribute to individual differences in fecal microbiome composition. RESULTS: Among 110 demographic, clinical, and environmental factors, 11 were identified as significantly correlated with α-diversity, ß-diversity, or abundance of specific microbial communities in multivariable models. Age and blood alanine aminotransferase levels showed the strongest associations with microbiome diversity. In total, all non-genetic factors explained 16.4% of the variance. We then searched for associations between > 5 million single nucleotide polymorphisms and the same indicators of fecal microbiome diversity, including the significant non-genetic factors as covariates. No genome-wide significant associations were identified after correction for multiple testing. A small fraction of previously reported associations between human genetic variants and specific taxa could be replicated in our cohort, while no replication was observed for any of the diversity metrics. CONCLUSION: In a well-characterized cohort of healthy individuals, we identified several non-genetic variables associated with fecal microbiome diversity. In contrast, host genetics only had a negligible influence. Demographic and environmental factors are thus the main contributors to fecal microbiome composition in healthy individuals. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01699893.

Commensal-specific T cell plasticity promotes rapid tissue adaptation to injury.

Barrier tissues are primary targets of environmental stressors and are home to the largest number of antigen-experienced lymphocytes in the body, including commensal-specific T cells. We found that skin-resident commensal-specific T cells harbor a paradoxical program characterized by a type 17 program associated with a poised type 2 state. Thus, in the context of injury and exposure to inflammatory mediators such as interleukin-18, these cells rapidly release type 2 cytokines, thereby acquiring contextual functions. Such acquisition of a type 2 effector program promotes tissue repair. Aberrant type 2 responses can also be unleashed in the context of local defects in immunoregulation. Thus, commensal-specific T cells co-opt tissue residency and cell-intrinsic flexibility as a means to promote both local immunity and tissue adaptation to injury.

Contextual control of skin immunity and inflammation by Corynebacterium.

How defined microbes influence the skin immune system remains poorly understood. Here we demonstrate that Corynebacteria, dominant members of the skin microbiota, promote a dramatic increase in the number and activation of a defined subset of γδ T cells. This effect is long-lasting, occurs independently of other microbes, and is, in part, mediated by interleukin (IL)-23. Under steady-state conditions, the impact of Corynebacterium is discrete and noninflammatory. However, when applied to the skin of a host fed a high-fat diet, Corynebacterium accolens alone promotes inflammation in an IL-23-dependent manner. Such effect is highly conserved among species of Corynebacterium and dependent on the expression of a dominant component of the cell envelope, mycolic acid. Our data uncover a mode of communication between the immune system and a dominant genus of the skin microbiota and reveal that the functional impact of canonical skin microbial determinants is contextually controlled by the inflammatory and metabolic state of the host.

The human skin microbiome.

Functioning as the exterior interface of the human body with the environment, skin acts as a physical barrier to prevent the invasion of foreign pathogens while providing a home to the commensal microbiota. The harsh physical landscape of skin, particularly the desiccated, nutrient-poor, acidic environment, also contributes to the adversity that pathogens face when colonizing human skin. Despite this, the skin is colonized by a diverse microbiota. In this Review, we describe amplicon and shotgun metagenomic DNA sequencing studies that have been used to assess the taxonomic diversity of microorganisms that are associated with skin from the kingdom to the strain level. We discuss recent insights into skin microbial communities, including their composition in health and disease, the dynamics between species and interactions with the immune system, with a focus on Propionibacterium acnes, Staphylococcus epidermidis and Staphylococcus aureus.

Non-classical Immunity Controls Microbiota Impact on Skin Immunity and Tissue Repair.

Mammalian barrier surfaces are constitutively colonized by numerous microorganisms. We explored how the microbiota was sensed by the immune system and the defining properties of such responses. Here, we show that a skin commensal can induce T cell responses in a manner that is restricted to non-classical MHC class I molecules. These responses are uncoupled from inflammation and highly distinct from pathogen-induced cells. Commensal-specific T cells express a defined gene signature that is characterized by expression of effector genes together with immunoregulatory and tissue-repair signatures. As such, non-classical MHCI-restricted commensal-specific immune responses not only promoted protection to pathogens, but also accelerated skin wound closure. Thus, the microbiota can induce a highly physiological and pleiotropic form of adaptive immunity that couples antimicrobial function with tissue repair. Our work also reveals that non-classical MHC class I molecules, an evolutionarily ancient arm of the immune system, can promote homeostatic immunity to the microbiota.

White Adipose Tissue Is a Reservoir for Memory T Cells and Promotes Protective Memory Responses to Infection.

White adipose tissue bridges body organs and plays a fundamental role in host metabolism. To what extent adipose tissue also contributes to immune surveillance and long-term protective defense remains largely unknown. Here, we have shown that at steady state, white adipose tissue contained abundant memory lymphocyte populations. After infection, white adipose tissue accumulated large numbers of pathogen-specific memory T cells, including tissue-resident cells. Memory T cells in white adipose tissue expressed a distinct metabolic profile, and white adipose tissue from previously infected mice was sufficient to protect uninfected mice from lethal pathogen challenge. Induction of recall responses within white adipose tissue was associated with the collapse of lipid metabolism in favor of antimicrobial responses. Our results suggest that white adipose tissue represents a memory T cell reservoir that provides potent and rapid effector memory responses, positioning this compartment as a potential major contributor to immunological memory.

Staphylococcus aureus and Staphylococcus epidermidis strain diversity underlying pediatric atopic dermatitis.

The heterogeneous course, severity, and treatment responses among patients with atopic dermatitis (AD; eczema) highlight the complexity of this multifactorial disease. Prior studies have used traditional typing methods on cultivated isolates or sequenced a bacterial marker gene to study the skin microbial communities of AD patients. Shotgun metagenomic sequence analysis provides much greater resolution, elucidating multiple levels of microbial community assembly ranging from kingdom to species and strain-level diversification. We analyzed microbial temporal dynamics from a cohort of pediatric AD patients sampled throughout the disease course. Species-level investigation of AD flares showed greater Staphylococcus aureus predominance in patients with more severe disease and Staphylococcus epidermidis predominance in patients with less severe disease. At the strain level, metagenomic sequencing analyses demonstrated clonal S. aureus strains in more severe patients and heterogeneous S. epidermidis strain communities in all patients. To investigate strain-level biological effects of S. aureus, we topically colonized mice with human strains isolated from AD patients and controls. This cutaneous colonization model demonstrated S. aureus strain-specific differences in eliciting skin inflammation and immune signatures characteristic of AD patients. Specifically, S. aureus isolates from AD patients with more severe flares induced epidermal thickening and expansion of cutaneous T helper 2 (TH2) and TH17 cells. Integrating high-resolution sequencing, culturing, and animal models demonstrated how functional differences of staphylococcal strains may contribute to the complexity of AD disease.

Temporal Stability of the Human Skin Microbiome.

Biogeography and individuality shape the structural and functional composition of the human skin microbiome. To explore these factors' contribution to skin microbial community stability, we generated metagenomic sequence data from longitudinal samples collected over months and years. Analyzing these samples using a multi-kingdom, reference-based approach, we found that despite the skin's exposure to the external environment, its bacterial, fungal, and viral communities were largely stable over time. Site, individuality, and phylogeny were all determinants of stability. Foot sites exhibited the most variability; individuals differed in stability; and transience was a particular characteristic of eukaryotic viruses, which showed little site-specificity in colonization. Strain and single-nucleotide variant-level analysis showed that individuals maintain, rather than reacquire, prevalent microbes from the environment. Longitudinal stability of skin microbial communities generates hypotheses about colonization resistance and empowers clinical studies exploring alterations observed in disease states.

Microbiota-Dependent Sequelae of Acute Infection Compromise Tissue-Specific Immunity.

Infections have been proposed as initiating factors for inflammatory disorders; however, identifying associations between defined infectious agents and the initiation of chronic disease has remained elusive. Here, we report that a single acute infection can have dramatic and long-term consequences for tissue-specific immunity. Following clearance of Yersinia pseudotuberculosis, sustained inflammation and associated lymphatic leakage in the mesenteric adipose tissue deviates migratory dendritic cells to the adipose compartment, thereby preventing their accumulation in the mesenteric lymph node. As a consequence, canonical mucosal immune functions, including tolerance and protective immunity, are persistently compromised. Post-resolution of infection, signals derived from the microbiota maintain inflammatory mesentery remodeling and consequently, transient ablation of the microbiota restores mucosal immunity. Our results indicate that persistent disruption of communication between tissues and the immune system following clearance of an acute infection represents an inflection point beyond which tissue homeostasis and immunity is compromised for the long-term. VIDEO ABSTRACT.

Bone-Marrow-Resident NK Cells Prime Monocytes for Regulatory Function during Infection.

Tissue-infiltrating Ly6C(hi) monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DCs) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals after tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function.

Integrating host gene expression and the microbiome to explore disease pathogenesis.

In a recent study, rich clinical assessment and longitudinal study design are combined with host gene expression and microbial sequencing analyses to develop a framework for exploring disease etiology and outcomes in the context of human inflammatory disease.See related article: http://dx.doi.org/10.1186/s13059-015-0637-x.