Faecal microbial transfer and complex carbohydrates mediate protection against COPD.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DESIGN: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation. RESULTS: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. CONCLUSION: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically.

IL-23 favours outgrowth of spondyloarthritis-associated pathobionts and suppresses host support for homeostatic microbiota.

OBJECTIVES: Certain gut bacterial families, including Bacteroidaceae, Porphyromonadaceae and Prevotellaceae, are increased in people suffering from spondyloarthropathy (SpA), a disease group associated with IL23R signalling variants. To understand the relationship between host interleukin (IL)-23 signalling and gut bacterial dysbiosis in SpA, we inhibited IL-23 in dysbiotic ZAP-70-mutant SKG mice that develop IL-23-dependent SpA-like arthritis, psoriasis-like skin inflammation and Crohn's-like ileitis in response to microbial beta 1,3-glucan (curdlan). METHODS: We treated SKG mice weekly with anti-IL-23 or isotype mAb for 3 weeks, rested them for 3 weeks, then administered curdlan or saline. We collected faecal samples longitudinally, assessed arthritis, spondylitis, psoriasis and ileitis histologically, and analysed the microbiota community profiles using next-generation sequencing. We used multivariate sparse partial least squares discriminant analysis to identify operational taxonomic unit (OTU) signatures best classifying treatment groups and linear regression to develop a predictive model of disease severity. RESULTS: IL-23p19 inhibition in naïve SKG mice decreased Bacteroidaceae, Porphyromonadaceae and Prevotellaceae. Abundance of Clostridiaceae and Lachnospiraceae families concomitantly increased, and curdlan-mediated SpA development decreased. Abundance of Enterobacteriaceae and Porphyromonadaceae family and reduction in Lachnospiraceae Dorea genus OTUs early in disease course were associated with disease severity in affected tissues. CONCLUSIONS: Dysbiosis in SKG mice reflects human SpA and is IL-23p19 dependent. In genetically susceptible hosts, IL-23p19 favours outgrowth of SpA-associated pathobionts and reduces support for homeostatic-inducing microbiota. The relative abundance of specific pathobionts is associated with disease severity.

Acute graft-versus-host disease is regulated by an IL-17-sensitive microbiome.

Donor T-cell-derived interleukin-17A (IL-17A) can mediate late immunopathology in graft-versus-host disease (GVHD), however protective roles remain unclear. Using multiple cytokine and cytokine receptor subunit knockout mice, we demonstrate that stem cell transplant recipients lacking the ability to generate or signal IL-17 develop intestinal hyper-acute GVHD. This protective effect is restricted to the molecular interaction of IL-17A and/or IL-17F with the IL-17 receptor A/C (IL-17RA/C). The protection from GVHD afforded by IL-17A required secretion from, and signaling in, both hematopoietic and nonhematopoietic host tissue. Given the intestinal-specificity of the disease in these animals, we cohoused wild-type (WT) with IL-17RA and IL-17RC-deficient mice, which dramatically enhanced the susceptibility of WT mice to acute GVHD. Furthermore, the gut microbiome of WT mice shifted toward that of the IL-17RA/C mice during cohousing prior to transplant, confirming that an IL-17-sensitive gut microbiota controls susceptibility to acute GVHD. Finally, induced IL-17A depletion peritransplant also enhanced acute GVHD, consistent with an additional protective role for this cytokine independent of effects on dysbiosis.

Reestablishment of recipient-associated microbiota in the lung allograft is linked to reduced risk of bronchiolitis obliterans syndrome.

RATIONALE: Bronchiolitis obliterans syndrome (BOS) is the primary limiting factor for long-term survival after lung transplantation, and has previously been associated with microbial infections. OBJECTIVES: To cross-sectionally and longitudinally characterize microbial communities in allografts from transplant recipients with and without BOS using a culture-independent method based on high-throughput sequencing. METHODS: Allografts were sampled by bronchoalveolar lavage, and microbial communities were profiled using 16S rRNA gene amplicon pyrosequencing. Community profiles were compared using the weighted Unifrac metric and the relationship between microbial populations, BOS, and other covariates was explored using PERMANOVA and logistic regression. MEASUREMENTS AND MAIN RESULTS: Microbial communities in transplant patients fell into two main groups: those dominated by Pseudomonas or those dominated by Streptococcus and Veillonella, which seem to be mutually exclusive lung microbiomes. Aspergillus culture was also negatively correlated with the Pseudomonas-dominated group. The reestablishment of dominant populations present in patients pretransplant, notably Pseudomonas in individuals with cystic fibrosis, was negatively correlated with BOS. CONCLUSIONS: Recolonization of the allograft by Pseudomonas in individuals with cystic fibrosis is not associated with BOS. In general, reestablishment of pretransplant lung populations in the allograft seems to have a protective effect against BOS, whereas de novo acquisition of microbial populations often belonging to the same genera may increase the risk of BOS.

Changes in the skin microbiome associated with squamous cell carcinoma in transplant recipients.

Actinic keratoses (AK) arise in severely photo-damaged skin and can progress to squamous cell carcinomas (SCC). AK and SCC are common in Caucasian populations, and immunosuppressed individuals have a markedly higher risk of developing SCC. An overabundance of Staphylococcus aureus has been reported in AK and SCC lesions of immunocompetent individuals, however, the AK/SCC microbiome in immunosuppressed cohorts has not been investigated. Here, the microbial profile and bacterial load of AK, SCC and control skin swabs from 32 immunosuppressed organ transplant recipients were characterised via SSU rRNA gene sequencing and qPCR, and compared to a previously described immunocompetent cohort. Although the taxonomic composition of skin swab samples was mostly subject-specific, significant differences were observed between control skin, AK, and SCC in both cohorts. Surface bacterial load was increased and alpha diversity decreased in AK and SCC compared to control skin due to an increased abundance of Staphylococcus species and relative decrease of skin commensals. Staphylococcus epidermidis predominated on SCC from transplant recipients in contrast to SCC of immunocompetent subjects dominated by S. aureus. In conclusion, AK and SCC of immunosuppressed and immunocompetent subjects present with distinctive microbial dysbioses, which may be relevant to SCC pathogenesis and progression.

Secreted Toxins From Staphylococcus aureus Strains Isolated From Keratinocyte Skin Cancers Mediate Pro-tumorigenic Inflammatory Responses in the Skin.

Squamous cell carcinoma (SCC) is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses (AK). Chronic inflammation is a well-known promoter of skin cancer progression. AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus (S. aureus). Certain secreted products from S. aureus are known to promote cutaneous pro-inflammatory responses; however, not all S. aureus strains produce these. As inflammation plays a key role in SCC development, we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S. aureus. Sterile culture supernatants ("secretomes") of S. aureus clinical strains isolated from AK and SCC were applied to human keratinocytes in vitro. Some S. aureus secretomes induced keratinocytes to overexpress inflammatory mediators that have been linked to skin carcinogenesis, including IL-6, IL-8, and TNFα. A large phenotypic variation between the tested clinical strains was observed. Strains that are highly pro-inflammatory in vitro also caused more pronounced skin inflammation in mice. Proteomic characterization of S. aureus secretomes using mass spectrometry established that specific S. aureus enzymes and cytolytic toxins, including hemolysins, phenol-soluble modulins, and serine proteases, as well as currently uncharacterized proteins, correlate with the pro-inflammatory S. aureus phenotype. This study is the first to describe the toxin secretion profiles of AK and SCC-associated S. aureus, and their potential to induce a pro-inflammatory environment in the skin. Further studies are needed to establish whether these S. aureus products promote SCC development by mediating chronic inflammation.

Continuous pre- and post-transplant exposure to a disease-associated gut microbiome promotes hyper-acute graft-versus-host disease in wild-type mice.

OBJECTIVE: The gut microbiome plays a key role in the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation. Here we investigate the individual contribution of the pre- and post-transplant gut microbiome to acute GVHD using a well-studied mouse model. DESIGN: Wild-type mice were cohoused with IL-17RA-/ - mice, susceptible to hyperacute GVHD, either pre- or post-transplant alone or continuously (i.e., pre- and post-transplant). Fecal samples were collected from both WT and IL-17RA-/ - mice pre- and post-cohousing and post-transplant and the microbiome analyzed using metagenomic sequencing. RESULTS: Priming wild-type mice via cohousing pre-transplant only is insufficient to accelerate GVHD, however, accelerated disease is observed in WT mice cohoused post-transplant only. When mice are cohoused continuously, the effect of priming and exacerbation is additive, resulting in a greater acceleration of disease in WT mice beyond that seen with cohousing post-transplant only. Metagenomic analysis of the microbiome revealed pre-transplant cohousing is associated with the transfer of specific species within two as-yet-uncultured genera of the bacterial family Muribaculaceae; CAG-485 and CAG-873. Post-transplant, we observed GVHD-associated blooms of Enterobacteriaceae members Escherichia coli and Enterobacter hormaechei subsp. steigerwaltii, and hyperacute GVHD gut microbiome distinct from that associated with delayed-onset disease (>10 days post-transplant). CONCLUSION: These results clarify the importance of the peri-transplant microbiome in the susceptibility to acute GVHD post-transplant and demonstrate the species-specific nature of this association.

Disease-associated gut microbiome and metabolome changes in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the third commonest cause of death globally, and manifests as a progressive inflammatory lung disease with no curative treatment. The lung microbiome contributes to COPD progression, but the function of the gut microbiome remains unclear. Here we examine the faecal microbiome and metabolome of COPD patients and healthy controls, finding 146 bacterial species differing between the two groups. Several species, including Streptococcus sp000187445, Streptococcus vestibularis and multiple members of the family Lachnospiraceae, also correlate with reduced lung function. Untargeted metabolomics identifies a COPD signature comprising 46% lipid, 20% xenobiotic and 20% amino acid related metabolites. Furthermore, we describe a disease-associated network connecting Streptococcus parasanguinis_B with COPD-associated metabolites, including N-acetylglutamate and its analogue N-carbamoylglutamate. While correlative, our results suggest that the faecal microbiome and metabolome of COPD patients are distinct from those of healthy individuals, and may thus aid in the search for biomarkers for COPD.

A Natural History of Actinic Keratosis and Cutaneous Squamous Cell Carcinoma Microbiomes.

Cutaneous squamous cell carcinoma (SCC) is the second-most-common cancer in Australia. The majority of SCCs progress from premalignant actinic keratosis (AK) lesions that form on chronically sun-exposed skin. The role of skin microbiota in this progression is not well understood; therefore, we performed a longitudinal microbiome analysis of AKs and SCCs using a cohort of 13 SCC-prone immunocompetent men. The majority of variability in microbial profiles was attributable to subject, followed by time and lesion type. Propionibacterium and Malassezia organisms were relatively more abundant in nonlesional photodamaged skin than in AKs and SCCs. Staphylococcus was most commonly associated with lesional skin, in particular, sequences most closely related to Staphylococcus aureus Of 11 S. aureus-like operational taxonomic units (OTUs), six were significantly associated with SCC lesions across seven subjects, suggesting their specific involvement with AK-to-SCC progression. If a causative link exists between certain S. aureus-like OTUs and SCC etiology, therapeutic approaches specifically targeting these bacteria could be used to reduce SCC.IMPORTANCE Actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) are two of the most common dermatologic conditions in Western countries and cause substantial morbidity worldwide. The role of human papillomaviruses under these conditions has been well studied yet remains inconclusive. One PCR-based study has investigated bacteria in the etiology of these conditions; however, no study has investigated the microbiomes of AK and SCC more broadly. We longitudinally profiled the microbiomes of 112 AK lesions, profiled cross sections of 32 spontaneously arising SCC lesions, and compared these to matching nonlesional photodamaged control skin sites. We identified commonly occurring strains of Propionibacterium and Malassezia at higher relative abundances on nonlesional skin than in AK and SCC lesions, and strains of Staphylococcus aureus were relatively more abundant in lesional than nonlesional skin. These findings may aid in the prevention of SCC.

Recipient mucosal-associated invariant T cells control GVHD within the colon.

Mucosal-associated invariant T (MAIT) cells are a unique innate-like T cell subset that responds to a wide array of bacteria and yeast through recognition of riboflavin metabolites presented by the MHC class I-like molecule MR1. Here, we demonstrate using MR1 tetramers that recipient MAIT cells are present in small but definable numbers in graft-versus-host disease (GVHD) target organs and protect from acute GVHD in the colon following bone marrow transplantation (BMT). Consistent with their preferential juxtaposition to microbial signals in the colon, recipient MAIT cells generate large amounts of IL-17A, promote gastrointestinal tract integrity, and limit the donor alloantigen presentation that in turn drives donor Th1 and Th17 expansion specifically in the colon after BMT. Allogeneic BMT recipients deficient in IL-17A also develop accelerated GVHD, suggesting MAIT cells likely regulate GVHD, at least in part, by the generation of this cytokine. Indeed, analysis of stool microbiota and colon tissue from IL-17A-/- and MR1-/- mice identified analogous shifts in microbiome operational taxonomic units (OTU) and mediators of barrier integrity that appear to represent pathways controlled by similar, IL-17A-dependent mechanisms. Thus, MAIT cells act to control barrier function to attenuate pathogenic T cell responses in the colon and, given their very high frequency in humans, likely represent an important population in clinical BMT.

Near complete genome sequence of the animal feed probiotic, Bacillus amyloliquefaciens H57.

Bacillus amyloliquefaciens H57 is a bacterium isolated from lucerne for its ability to prevent feed spoilage. Further interest developed when ruminants fed with H57-inoculated hay showed increased weight gain and nitrogen retention relative to controls, suggesting a probiotic effect. The near complete genome of H57 is ~3.96 Mb comprising 16 contigs. Within the genome there are 3,836 protein coding genes, an estimated sixteen rRNA genes and 69 tRNA genes. H57 has the potential to synthesise four different lipopeptides and four polyketide compounds, which are known antimicrobials. This antimicrobial capacity may facilitate the observed probiotic effect.

Genomic characterization of the uncultured Bacteroidales family S24-7 inhabiting the guts of homeothermic animals.

BACKGROUND: Our view of host-associated microbiota remains incomplete due to the presence of as yet uncultured constituents. The Bacteroidales family S24-7 is a prominent example of one of these groups. Marker gene surveys indicate that members of this family are highly localized to the gastrointestinal tracts of homeothermic animals and are increasingly being recognized as a numerically predominant member of the gut microbiota; however, little is known about the nature of their interactions with the host. RESULTS: Here, we provide the first whole genome exploration of this family, for which we propose the name "Candidatus Homeothermaceae," using 30 population genomes extracted from fecal samples of four different animal hosts: human, mouse, koala, and guinea pig. We infer the core metabolism of "Ca. Homeothermaceae" to be that of fermentative or nanaerobic bacteria, resembling that of related Bacteroidales families. In addition, we describe three trophic guilds within the family, plant glycan (hemicellulose and pectin), host glycan, and α-glucan, each broadly defined by increased abundance of enzymes involved in the degradation of particular carbohydrates. CONCLUSIONS: "Ca. Homeothermaceae" representatives constitute a substantial component of the murine gut microbiota, as well as being present within the human gut, and this study provides important first insights into the nature of their residency. The presence of trophic guilds within the family indicates the potential for niche partitioning and specific roles for each guild in gut health and dysbiosis.

In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery.

Over the past decade, technological advances in whole genome amplification, microfluidics, flow sorting, and high-throughput sequencing have led to the development of single-cell genomics. Single-cell genomic approaches are typically applied to anonymous microbial cells with only morphology providing clues to their identity. However, targeted separation of microorganisms based on phylogenetic markers, such as the 16S rRNA gene, is beginning to emerge in the single-cell genomics field. Here, we describe an in-solution fluorescence in situ hybridization (FISH) protocol which can be combined with fluorescence-activated cell sorting (FACS) for separation of single cells or populations of interest from environmental samples. Sequencing of DNA obtained from sorted cells can be used for the recovery of draft quality genomes, and when performed in parallel with deep metagenomics, can be used to validate and further scaffold metagenomic assemblies. We illustrate in this chapter the feasibility of this FISH-FACS approach by describing the targeted recovery of a novel anaerobic methanotrophic archaeon.