Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine.

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

Factors Affecting the Gut Microbiome in Pediatric Intestinal Failure.

BACKGROUND: There is little data on gut microbiome and various factors that lead to dysbiosis in pediatric intestinal failure (PIF). This study aimed to characterize gut microbiome in PIF and determine factors that may affect microbial composition in these patients. METHODS: This is a single-center, prospective cohort study of children with PIF followed at our intestinal rehabilitation program. Stool samples were collected longitudinally at regular intervals over a 1-year period. Medical records were reviewed, and demographic and clinical data were collected. Medication history including the use of acid blockers, scheduled prophylactic antibiotics, and bile acid sequestrants was obtained. Gut microbial diversity among patients was assessed and compared according to various host characteristics of interest. RESULTS: The final analysis included 74 specimens from 12 subjects. Scheduled prophylactic antibiotics, presence of central line associated bloodstream infection (CLABSI) at the time of specimen collection, use of acid blockers, and ≥50% calories delivered via parenteral nutrition (PN) was associated with reduced alpha diversity, whereas increasing age was associated with improved alpha diversity at various microbial levels ( P value <0.05). Beta diversity differed with age, presence of CLABSI, use of scheduled antibiotics, acid blockers, percent calories via PN, and presence of oral feeds at various microbial levels ( P value <0.05). Single taxon analysis identified several taxa at several microbial levels, which were significantly associated with various host characteristics. CONCLUSION: Gut microbial diversity in PIF subjects is influenced by various factors involved in the rehabilitation process including medications, percent calories received parenterally, CLABSI events, the degree of oral feeding, and age. Additional investigation performed across multiple centers is needed to further understand the impact of these findings on important clinical outcomes in PIF.

Scheduled Empiric Antibiotics May Alter the Gut Microbiome and Nutrition Outcomes in Pediatric Intestinal Failure.

BACKGROUND: In this study, we aim to determine the effect of scheduled antibiotics on gut microbiome in pediatric intestinal failure (IF) and to evaluate the effect of the gut microbiome on nutrition outcomes in IF. METHODS: Fecal samples were collected at regular intervals from pediatric patients with IF for gut microbiome comparison between 2 cohorts: (group 1) those on scheduled prophylactic antibiotics and (group 2) those who were not on scheduled antibiotics. Gut microbiome composition and diversity were compared among the 2 cohorts. The association among gut microbiome composition, diversity, and nutrition outcomes (mainly ability to decrease parenteral nutrition [PN] energy requirement and ability to attain positive growth) was also determined. RESULTS: The microbiome of patients with IF on scheduled antibiotics differed significantly from those not on scheduled antibiotics. Abundance of certain Gram-negative and pathogenic bacteria (Pseudomonas, Prevotella, and Sutterella) was higher in the scheduled cohort. Patients with decreased Enterobacteriaceae demonstrated a greater ability to demonstrate a reduction in PN requirement, as well as attain positive growth. CONCLUSION: Scheduled antibiotics may alter the gut microbiome in children IF, which in turn may have an influence on important nutrition outcomes in pediatric IF. Further larger, multicenter studies are needed to determine the effect of scheduled antibiotics on the gut microbiome in this patient population and their overall effect on nutrition outcomes.

Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure.

Calorie-dense obesogenic diet (OBD) is a prime risk factor for cardiovascular disease in aging. However, increasing age coupled with changes in the diet can affect the interaction of intestinal microbiota influencing the immune system, which can lead to chronic inflammation. How age and calorie-enriched OBD interact with microbial flora and impact leukocyte profiling is currently under investigated. Here, we tested the interorgan hypothesis to determine whether OBD in young and aging mice alters the gut microbe composition and the splenic leukocyte profile in acute heart failure (HF). Young (2-mo-old) and aging (18-mo-old) mice were supplemented with standard diet (STD, ∼4% safflower oil diet) and OBD (10% safflower oil) for 2 mo and then subjected to coronary artery ligation to induce myocardial infarction. Fecal samples were collected pre- and post-diet intervention, and the microbial flora were analyzed using 16S variable region 4 rRNA gene DNA sequencing and Quantitative Insights Into Microbial Ecology informatics. The STD and OBD in aging mice resulted in an expansion of the genus Allobaculum in the fecal microbiota. However, we found a pathologic change in the neutrophil:lymphocyte ratio in aging mice in comparison with their young counterparts. Thus, calorie-enriched OBD dysregulated splenic leukocytes by decreasing immune-responsive F4/80+ and CD169+ macrophages in aging mice. OBD programmed neutrophil swarming with an increase in isoprostanoid levels, with dysregulation of lipoxygenases, cytokines, and metabolite-sensing receptor expression. In summary, calorie-dense OBD in aging mice disrupted the composition of the gut microbiome, which correlates with the development of integrative and system-wide nonresolving inflammation in acute HF.-Kain, V., Van Der Pol, W., Mariappan, N., Ahmad, A., Eipers, P., Gibson, D. L., Gladine, C., Vigor, C., Durand, T., Morrow, C., Halade, G. V. Obesogenic diet in aging mice disrupts gut microbe composition and alters neutrophil:lymphocyte ratio, leading to inflamed milieu in acute heart failure.

Composition and richness of the serum microbiome differ by age and link to systemic inflammation.

Advanced age has been associated with alterations to the microbiome within the intestinal tract as well as intestinal permeability (i.e., "leaky gut"). Prior studies suggest that intestinal permeability may contribute to increases in systemic inflammation-an aging hallmark-possibly via microorganisms entering the circulation. Yet, no studies exist describing the state of the circulating microbiome among older persons. To compare microbiota profiles in serum between healthy young (20-35 years, n = 24) and older adults (60-75 years, n = 24) as well as associations between differential microbial populations and prominent indices of age-related inflammation. Unweighted Unifrac analysis, a measure of ß-diversity, revealed that microbial communities clustered differently between young and older adults. Several measures of α-diversity, including chao1 (p = 0.001), observed species (p = 0.001), and phylogenetic diversity (p = 0.002) differed between young and older adults. After correction for false discovery rate (FDR), age groups differed (all p values ≤ 0.016) in the relative abundance of the phyla Bacteroidetes, SR1, Spirochaetes, Bacteria_Other, TM7, and Tenericutes. Significant positive correlations (p values ≤ 0.017 after FDR correction) were observed between IGF1 and Bacteroidetes (ρ = 0.380), Spirochaetes (ρ = 0.528), SR1 (ρ = 0.410), and TM7 (ρ = 0.399). Significant inverse correlations were observed for IL6 with Bacteroidetes (ρ = - 0.398) and TM7 (ρ = - 0.423), as well as for TNFα with Bacteroidetes (ρ = - 0.344). Similar findings were observed at the class taxon. These data are the first to demonstrate that the richness and composition of the serum microbiome differ between young and older adults and that these factors are linked to indices of age-related inflammation.

Erratum: Author Correction: Identification of donor microbe species that colonize and persist long term in the recipient after fecal transplant for recurrent Clostridium difficile.

[This corrects the article DOI: 10.1038/s41522-017-0020-7.].

Distal airway microbiome is associated with immunoregulatory myeloid cell responses in lung transplant recipients.

BACKGROUND: Long-term survival of lung transplant recipients (LTRs) is limited by the occurrence of bronchiolitis obliterans syndrome (BOS). Recent evidence suggests a role for microbiome alterations in the occurrence of BOS, although the precise mechanisms are unclear. In this study we evaluated the relationship between the airway microbiome and distinct subsets of immunoregulatory myeloid-derived suppressor cells (MDSCs) in LTRs. METHODS: Bronchoalveolar lavage (BAL) and simultaneous oral wash and nasal swab samples were collected from adult LTRs. Microbial genomic DNA was isolated, 16S rRNA genes amplified using V4 primers, and polymerase chain reaction (PCR) products sequenced and analyzed. BAL MDSC subsets were enumerated using flow cytometry. RESULTS: The oral microbiome signature differs from that of the nasal, proximal and distal airway microbiomes, whereas the nasal microbiome is closer to the airway microbiome. Proximal and distal airway microbiome signatures of individual subjects are distinct. We identified phenotypic subsets of MDSCs in BAL, with a higher proportion of immunosuppressive MDSCs in the proximal airways, in contrast to a preponderance of pro-inflammatory MDSCs in distal airways. Relative abundance of distinct bacterial phyla in proximal and distal airways correlated with particular airway MDSCs. Expression of CCAAT/enhancer binding protein (C/EBP)-homologous protein (CHOP), an endoplasmic (ER) stress sensor, was increased in immunosuppressive MDSCs when compared with pro-inflammatory MDSCs. CONCLUSIONS: The nasal microbiome closely resembles the microbiome of the proximal and distal airways in LTRs. The association of distinct microbial communities with airway MDSCs suggests a functional relationship between the local microbiome and MDSC phenotype, which may contribute to the pathogenesis of BOS.

Comparison of two bioinformatics tools used to characterize the microbial diversity and predictive functional attributes of microbial mats from Lake Obersee, Antarctica.

In this study, using NextGen sequencing of the collective 16S rRNA genes obtained from two sets of samples collected from Lake Obersee, Antarctica, we compared and contrasted two bioinformatics tools, PICRUSt and Tax4Fun. We then developed an R script to assess the taxonomic and predictive functional profiles of the microbial communities within the samples. Taxa such as Pseudoxanthomonas, Planctomycetaceae, Cyanobacteria Subsection III, Nitrosomonadaceae, Leptothrix, and Rhodobacter were exclusively identified by Tax4Fun that uses SILVA database; whereas PICRUSt that uses Greengenes database uniquely identified Pirellulaceae, Gemmatimonadetes A1-B1, Pseudanabaena, Salinibacterium and Sinobacteraceae. Predictive functional profiling of the microbial communities using Tax4Fun and PICRUSt separately revealed common metabolic capabilities, while also showing specific functional IDs not shared between the two approaches. Combining these functional predictions using a customized R script revealed a more inclusive metabolic profile, such as hydrolases, oxidoreductases, transferases; enzymes involved in carbohydrate and amino acid metabolisms; and membrane transport proteins known for nutrient uptake from the surrounding environment. Our results present the first molecular-phylogenetic characterization and predictive functional profiles of the microbial mat communities in Lake Obersee, while demonstrating the efficacy of combining both the taxonomic assignment information and functional IDs using the R script created in this study for a more streamlined evaluation of predictive functional profiles of microbial communities.

Identification of donor microbe species that colonize and persist long term in the recipient after fecal transplant for recurrent Clostridium difficile.

Fecal microbiota transplantation has been shown to be an effective treatment for patients with recurrent C. difficile colitis. Although fecal microbiota transplantation helps to re-establish a normal gut function in patients, the extent of the repopulation of the recipient microbial community varies. To further understand this variation, it is important to determine the fate of donor microbes in the patients following fecal microbiota transplantation. We have developed a new method that utilizes the unique single nucleotide variants of gut microbes to accurately identify microbes in paired fecal samples from the same individual taken at different times. Using this method, we identified transplant donor microbes in seven recipients 3-6 months after fecal microbiota transplantation; in two of these fecal microbiota transplantation, we were able to identify donor microbes that persist in recipients up to 2 years post-fecal microbiota transplantation. Our study provides new insights into the dynamics of the reconstitution of the gastrointestinal microbe community structure following fecal microbiota transplantation.

Metagenomics approach to the study of the gut microbiome structure and function in zebrafish Danio rerio fed with gluten formulated diet.

In this study, we report the gut microbial composition and predictive functional profiles of zebrafish, Danio rerio, fed with a control formulated diet (CFD), and a gluten formulated diet (GFD) using a metagenomics approach and bioinformatics tools. The microbial communities of the GFD-fed D. rerio displayed heightened abundances of Legionellales, Rhizobiaceae, and Rhodobacter, as compared to the CFD-fed counterparts. Predicted metagenomics of microbial communities (PICRUSt) in GFD-fed D. rerio showed KEGG functional categories corresponding to bile secretion, secondary bile acid biosynthesis, and the metabolism of glycine, serine, and threonine. The CFD-fed D. rerio exhibited KEGG functional categories of bacteria-mediated cobalamin biosynthesis, which was supported by the presence of cobalamin synthesizers such as Bacteroides and Lactobacillus. Though these bacteria were absent in GFD-fed D. rerio, a comparable level of the cobalamin biosynthesis KEGG functional category was observed, which could be contributed by the compensatory enrichment of Cetobacterium. Based on these results, we conclude D. rerio to be a suitable alternative animal model for the use of a targeted metagenomics approach along with bioinformatics tools to further investigate the relationship between the gluten diet and microbiome profile in the gut ecosystem leading to gastrointestinal diseases and other undesired adverse health effects.

The Airway Microbiome at Birth.

Alterations of pulmonary microbiome have been recognized in multiple respiratory disorders. It is critically important to ascertain if an airway microbiome exists at birth and if so, whether it is associated with subsequent lung disease. We found an established diverse and similar airway microbiome at birth in both preterm and term infants, which was more diverse and different from that of older preterm infants with established chronic lung disease (bronchopulmonary dysplasia). Consistent temporal dysbiotic changes in the airway microbiome were seen from birth to the development of bronchopulmonary dysplasia in extremely preterm infants. Genus Lactobacillus was decreased at birth in infants with chorioamnionitis and in preterm infants who subsequently went on to develop lung disease. Our results, taken together with previous literature indicating a placental and amniotic fluid microbiome, suggest fetal acquisition of an airway microbiome. We speculate that the early airway microbiome may prime the developing pulmonary immune system, and dysbiosis in its development may set the stage for subsequent lung disease.

Colonization potential to reconstitute a microbe community in patients detected early after fecal microbe transplant for recurrent C. difficile.

BACKGROUND: Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient's gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice. RESULTS: A total of nine samples from three human donors, recipient's pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01%). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8%, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55%, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2-4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7% of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10%. CONCLUSION: We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.

Getting started with microbiome analysis: sample acquisition to bioinformatics.

Historically, in order to study microbes, it was necessary to grow them in the laboratory. It was clear though that many microbe communities were refractory to study because none of the members could be grown outside of their native habitat. The development of culture-independent methods to study microbiota using high-throughput sequencing of the 16S ribosomal RNA gene variable regions present in all prokaryotic organisms has provided new opportunities to investigate complex microbial communities. In this unit, the process for a microbiome analysis is described. Many of the components required for this process may already exist. A pipeline is described for acquisition of samples from different sites on the human body, isolation of microbial DNA, and DNA sequencing using the Illumina MiSeq sequencing platform. Finally, a new analytical workflow for basic bioinformatics data analysis, QWRAP, is described, which can be used by clinical and basic science investigators.

HIV gene expression from intact proviruses positioned in bacterial artificial chromosomes at integration sites previously identified in latently infected T cells.

HIV integration predominantly occurs in introns of transcriptionally active genes. To study the impact of the integration site on HIV gene expression, a complete HIV-1 provirus (with GFP as a fusion with Nef) was inserted into bacterial artificial chromosomes (BACs) at three sites previously identified in latent T cells of patients: topoisomerase II (Top2A), DNA methyltransferase 1 (DNMT1), or basic leucine transcription factor 2 (BACH2). Transfection of BAC-HIV into 293T cells resulted in a fourfold difference in production of infectious HIV-1. Cell lines were established that contained BAC-Top2A, BAC-DNMT1, or BAC-BACH2, but only BAC-DNMT1 spontaneously produced virus, albeit at a low level. Stimulation with TNF-α resulted in virus production from four of five BAC-Top2A and all BAC-DNMT1 cell lines, but not from the BAC-BACH2 lines. The results of these studies highlight differences between integration sites identified in latent T cells to support virus production and reactivation from latency.

tRNA isoacceptor preference prior to retrovirus Gag-Pol junction links primer selection and viral translation.

An essential step in the replication of all retroviruses is the capture of a cellular tRNA that is used as the primer for reverse transcription. The 3'-terminal 18 nucleotides of the tRNA are complementary to the primer binding site (PBS). Moloney murine leukemia virus (MuLV) preferentially captures tRNA(Pro). To investigate the specificity of primer selection, the PBS of MuLV was altered to be complementary to different tRNAs. Analysis of the infectivity of the virus and stability of the PBS following in vitro replication revealed that MuLV prefers to select tRNA(Pro), tRNA(Gly), or tRNA(Arg). Previous studies from our laboratory have suggested that tRNA primer capture is coordinated with translation. Coincidentally, a cluster of proline, arginine, and glycine precedes the Gag-Pol junction of MuLV. Human immunodeficiency virus type 1 (HIV-1), which prefers tRNA(3)(Lys) as the primer, can be forced to utilize tRNA(Met), tRNA(1,2)(Lys), tRNA(His), or tRNA(Glu), although these viruses replicate poorly. Codons for methionine, lysine, histidine, or glutamic acid are found prior to the Gag-Pol frameshift site. HIV-1 was mutated so that the 5 lysine codons prior to the Gag-Pol frameshift region were specific for tRNA(1,2)(Lys). HIV-1 forced to use tRNA(1,2)(Lys) as the primer, with the mutation of codons specific for tRNA(1,2)(Lys) prior to the Gag-Pol junction, had enhanced infectivity and replicated similarly to the wild-type virus. The results demonstrate that codon preference prior to the Gag-Pol junction influences primer selection and suggest a coordination of Gag-Pol synthesis and acquisition of the tRNA primer required for retrovirus replication.

HIV-1 designed to use different tRNAGln isoacceptors prefers to select tRNAThr for replication.

BACKGROUND: Previous studies have shown that infection with human immunodeficiency virus type 1 (HIV-1) causes acceleration of the synthesis of glutamine tRNA (tRNAGln) in infected cells. To investigate whether this might influence HIV-1 to utilize tRNAGln as a primer for initiation of reverse transcription, we have constructed HIV-1 proviral genomes in which the PBS and the A-loop region upstream of the PBS have been made complementary to either the anticodon region of tRNAGln,1 or tRNAGln,3 and 3' terminal 18 nucleotides of each isoacceptor of tRNAGln. RESULTS: Viruses in which the PBS was altered to be complementary to tRNAGln,1 or tRNAGln,3 with or without the A-loop all exhibited a lower infectivity than the wild type virus. Viruses with only the PBS complementary to tRNAGln,1 or tRNAGln,3 reverted to wild type following culture in SupT1 cells. Surprisingly, viruses in which the PBS and A-loop were complementary to tRNAGln,1 did not grow in SupT1 cells, while viruses in which the PBS and A-loop were made complementary to tRNAGln,3 grew slowly in SupT1 cells. Analysis of the PBS of this virus revealed that it had reverted to select tRNAThr as the primer, which shares complementarity in 15 of 18 nucleotides with the PBS complementary to tRNAGln,3. CONCLUSION: The results of these studies support the concept that the HIV-1 has preferred tRNAs that can be selected as primers for replication.