Adipose tissue derived bacteria are associated with inflammation in obesity and type 2 diabetes

Objective Bacterial translocation to various organs including human adipose tissue (AT) due to increased intestinal permeability remains poorly understood. We hypothesised that: (1) bacterial presence is highly tissue specific and (2) related in composition and quantity to immune inflammatory and metabolic burden. Design We quantified and sequenced the bacterial 16S rRNA gene in blood and AT samples (omental, mesenteric and subcutaneous) of 75 subjects with obesity with or without type 2 diabetes (T2D) and used catalysed reporter deposition (CARD) – fluorescence in situ hybridisation (FISH) to detect bacteria in AT. Results Under stringent experimental and bioinformatic control for contaminants, bacterial DNA was detected in blood and omental, subcutaneous and mesenteric AT samples in the range of 0.1 to 5 pg/µg DNA isolate. Moreover, CARD-FISH allowed the detection of living, AT-borne bacteria. Proteobacteria and Firmicutes were the predominant phyla, and bacterial quantity was associated with immune cell infiltration, inflammatory and metabolic parameters in a tissue-specific manner. Bacterial composition differed between subjects with and without T2D and was associated with related clinical measures, including systemic and tissues-specific inflammatory markers. Finally, treatment of adipocytes with bacterial DNA in vitro stimulated the expression of TNFA and IL6. Conclusions Our study provides contaminant aware evidence for the presence of bacteria and bacterial DNA in several ATs in obesity and T2D and suggests an important role of bacteria in initiating and sustaining local AT subclinical inflammation and therefore impacting metabolic sequelae of obesity.

[1]  Pedro M. Valero-Mora,et al.  ggplot2: Elegant Graphics for Data Analysis , 2010 .

[2]  Susan Holmes,et al.  phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data , 2013, PloS one.

[3]  G. Bergström,et al.  Intestinal Ralstonia pickettii augments glucose intolerance in obesity , 2017, PloS one.

[4]  W. D. de Vos,et al.  A purified membrane protein from Akkermansia muciniphila or the pasteurized bacterium improves metabolism in obese and diabetic mice , 2016, Nature Medicine.

[5]  J. Fine,et al.  ON THE ABSORPTION OF BACTERIAL ENDOTOXIN FROM THE GASTRO-INTESTINAL TRACT OF THE NORMAL AND SHOCKED ANIMAL , 1960, The Journal of experimental medicine.

[6]  C. Consolandi,et al.  Adipose tissue microbiota in humans: an open issue , 2016, International Journal of Obesity.

[7]  M. Robertson,et al.  Increased intestinal permeability to oral chromium (51Cr) ‐EDTA in human Type 2 diabetes , 2014, Diabetic medicine : a journal of the British Diabetic Association.

[8]  Francesco Asnicar,et al.  Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2 , 2019, Nature Biotechnology.

[9]  S Kumar,et al.  Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes. , 2007, American journal of physiology. Endocrinology and metabolism.

[10]  K. Clément,et al.  Increased jejunal permeability in human obesity is revealed by a lipid challenge and is linked to inflammation and type 2 diabetes , 2018, The Journal of pathology.

[11]  J. T. Curtis,et al.  An Ordination of the Upland Forest Communities of Southern Wisconsin , 1957 .

[12]  Hadley Wickham,et al.  ggplot2 - Elegant Graphics for Data Analysis (2nd Edition) , 2017 .

[13]  M. Membrez,et al.  Gut microbiota modulation with norfloxacin and ampicillin enhances glucose tolerance in mice , 2008, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  Jian Xu,et al.  Impact of DNA extraction method and targeted 16S-rRNA hypervariable region on oral microbiota profiling , 2018, Scientific Reports.

[15]  Bingxuan Wang,et al.  Leptin- and Leptin Receptor-Deficient Rodent Models: Relevance for Human Type 2 Diabetes , 2014, Current diabetes reviews.

[16]  P. Sansonetti,et al.  Intestinal mucosal adherence and translocation of commensal bacteria at the early onset of type 2 diabetes: molecular mechanisms and probiotic treatment , 2011, EMBO molecular medicine.

[17]  Roland Eils,et al.  circlize implements and enhances circular visualization in R , 2014, Bioinform..

[18]  Benjamin Geiger,et al.  Hyperglycemia drives intestinal barrier dysfunction and risk for enteric infection , 2018, Science.

[19]  S. Simpson.  Of Mice . . . , 2004, Science.

[20]  E. Mardis,et al.  An obesity-associated gut microbiome with increased capacity for energy harvest , 2006, Nature.

[21]  Benjamin D. Kaehler,et al.  Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin , 2018, Microbiome.

[22]  W. Yam,et al.  Microbiology of Odontogenic Bacteremia: beyond Endocarditis , 2009, Clinical Microbiology Reviews.

[23]  M. Surette,et al.  Defective NOD2 peptidoglycan sensing promotes diet-induced inflammation, dysbiosis, and insulin resistance , 2015, EMBO molecular medicine.

[24]  C. Apovian,et al.  Inducible Toll‐like Receptor and NF‐κB Regulatory Pathway Expression in Human Adipose Tissue , 2008, Obesity.

[25]  D. Cowan,et al.  The Healthy Human Blood Microbiome: Fact or Fiction? , 2019, Front. Cell. Infect. Microbiol..

[26]  K. Katoh,et al.  MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. , 2002, Nucleic acids research.

[27]  D. Relman,et al.  Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data , 2017, Microbiome.

[28]  Julian Parkhill,et al.  Recognizing the reagent microbiome , 2018, Nature Microbiology.

[29]  R. Bibiloni,et al.  Changes in Gut Microbiota Control Metabolic Endotoxemia-Induced Inflammation in High-Fat Diet–Induced Obesity and Diabetes in Mice , 2008, Diabetes.

[30]  Yuko Sakurai,et al.  Gut Dysbiosis and Detection of “Live Gut Bacteria” in Blood of Japanese Patients With Type 2 Diabetes , 2014, Diabetes Care.

[31]  M. Stumvoll,et al.  Widely Used Commercial ELISA Does Not Detect Precursor of Haptoglobin2, but Recognizes Properdin as a Potential Second Member of the Zonulin Family , 2018, Front. Endocrinol..

[32]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[33]  Mathieu Almeida,et al.  Dietary intervention impact on gut microbial gene richness , 2013, Nature.

[34]  M. Surette,et al.  Type 2 diabetes influences bacterial tissue compartmentalisation in human obesity , 2020, Nature Metabolism.

[35]  Marcus J. Claesson,et al.  Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions , 2010, Nucleic acids research.

[36]  J. Bressan,et al.  Intestinal permeability parameters in obese patients are correlated with metabolic syndrome risk factors. , 2012, Clinical nutrition.

[37]  M. Martínez‐Porchas,et al.  Significant loss of sensitivity and specificity in the taxonomic classification occurs when short 16S rRNA gene sequences are used , 2016, Heliyon.

[38]  Paul J. McMurdie,et al.  DADA2: High resolution sample inference from Illumina amplicon data , 2016, Nature Methods.

[39]  B. Fagerberg,et al.  Intestinal Permeability Is Associated With Visceral Adiposity in Healthy Women , 2011, Obesity.

[40]  Ian D. Caterson,et al.  Increased Gut Permeability and Microbiota Change Associate with Mesenteric Fat Inflammation and Metabolic Dysfunction in Diet-Induced Obese Mice , 2012, PloS one.

[41]  Pelin Yilmaz,et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools , 2012, Nucleic Acids Res..

[42]  B. Balkau,et al.  Blood Microbiota Dysbiosis Is Associated with the Onset of Cardiovascular Events in a Large General Population: The D.E.S.I.R. Study , 2013, PloS one.

[43]  F. Servant,et al.  Changes in blood microbiota profiles associated with liver fibrosis in obese patients: A pilot analysis , 2016, Hepatology.

[44]  K. Rudi,et al.  Plasma Lipopolysaccharide Is Closely Associated With Glycemic Control and Abdominal Obesity , 2013, Diabetes Care.

[45]  J. Clemente,et al.  Gut Microbiota from Twins Discordant for Obesity Modulate Metabolism in Mice , 2013, Science.

[46]  P. Brun,et al.  Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis. , 2007, American journal of physiology. Gastrointestinal and liver physiology.

[47]  R. Knight,et al.  The Effect of Diet on the Human Gut Microbiome: A Metagenomic Analysis in Humanized Gnotobiotic Mice , 2009, Science Translational Medicine.

[48]  R. Amann,et al.  Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations , 1990, Applied and environmental microbiology.

[49]  Karsten Zengler,et al.  The microbiome extends to subepidermal compartments of normal skin , 2012, Nature Communications.

[50]  R. Irizarry.  ggplot2 , 2019, Introduction to Data Science.

[51]  W. D. de Vos,et al.  Pasteurized Akkermansia muciniphila increases whole-body energy expenditure and fecal energy excretion in diet-induced obese mice , 2020, Gut microbes.

[52]  C. Jobin,et al.  High-Fat Diet: Bacteria Interactions Promote Intestinal Inflammation Which Precedes and Correlates with Obesity and Insulin Resistance in Mouse , 2010, PloS one.

[53]  James R. Cole,et al.  Ribosomal Database Project: data and tools for high throughput rRNA analysis , 2013, Nucleic Acids Res..

[54]  M. Pfaffl,et al.  A new mathematical model for relative quantification in real-time RT-PCR. , 2001, Nucleic acids research.

[55]  R. Amann,et al.  Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. , 1993, Cytometry.

[56]  William A. Walters,et al.  Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys , 2015, mSystems.

[57]  P. Bork,et al.  Richness of human gut microbiome correlates with metabolic markers , 2013, Nature.

[58]  F. Crea,et al.  Epicardial adipose tissue microbial colonization and inflammasome activation in acute coronary syndrome. , 2017, International journal of cardiology.

[59]  M. Buck,et al.  Blood Microbiome Quantity and the Hyperdynamic Circulation in Decompensated Cirrhotic Patients , 2017, PloS one.

[60]  M. Kilian,et al.  Incidence of bacteremia after chewing, tooth brushing and scaling in individuals with periodontal inflammation. , 2006, Journal of clinical periodontology.

[61]  H. Tilg,et al.  Intestinal permeability – a new target for disease prevention and therapy , 2014, BMC Gastroenterology.

[62]  R. Burcelin,et al.  Metagenome and metabolism: the tissue microbiota hypothesis , 2013, Diabetes, obesity & metabolism.

[63]  K. Schleifer,et al.  The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. , 1999, Systematic and applied microbiology.