Human Microbiota Influence the Response to Anti-PD-1 in a Pre-clinical Model of Glioblastoma
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Human Microbiota Influence the Response to Anti-PD-1 in a Pre-clinical Model of Glioblastoma Kory J. Dees,1 Hyunmin Koo,2 J. Fraser Humphreys,3 Joseph A. Hakim,4 David K. Crossman,2 Michael R. Crowley,2 L. Burton Nabors,3 Etty N. Benveniste,1 Casey D. Morrow,1 and Braden C. McFarland1 1Department of Cell, Developmental and Integrative Biology, 2Department of Genetics, 3Department of Neurology, and 4School of Medicine, University of Alabama at Birmingham, Birmingham, AL The composition of the gut microbiome has been shown to determine responsiveness or resistance to immune checkpoint inhibitors (anti-PD-1) in patients with melanoma and other cancers. Unfortunately, although immunotherapy works well in glioblastoma (GBM) pre-clinical mouse models, the therapy has not demonstrated efficacy in humans. Most pre-clinical cancer studies have been done in mouse models using mouse gut microbiomes, but there are significant differences between mouse and human microbial gut compositions. To address this anomaly, we developed a novel humanized microbiome (HuM) model to study the response to immunotherapy in a pre-clinical mouse model of GBM. We used five healthy human donors for fecal transplantation of gnotobiotic mice. After the transplanted microbiomes stabilized, the mice were bred to generate five independent humanized mouse lines (HuM1-HuM5). Analysis of shotgun metagenomic sequencing data from fecal samples revealed a unique microbiome with significant differences in diversity and microbial composition among HuM1-HuM5 lines. Interestingly, we found that the HuM lines responded differently to the immune checkpoint inhibitor anti-PD-1. Specifically, we demonstrate that HuM2 and HuM3 mice are responsive to anti-PD-1 and displayed significantly increased survival compared to isotype controls, while HuM1, HuM4, and HuM5 mice are resistant to anti-PD-1. The question still remains of whether the “responsive” microbial communities in HuM2 and HuM3 can be therapeutically exploited and applicable in other tumor models, or if the “resistant” microbial communities in HuM1, HuM4, and HuM5 can be depleted and/or replaced. Future studies will assess responder microbial transplants as a method of enhancing immunotherapy.
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Therapeutic properties of Haladaptatus sp. strain R4
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Therapeutic properties of Haladaptatus sp. strain R4 Urmimala Sen, Subhra Kanti Mukhopadhyay Department of Microbiology, The University of Burdwan, Burdwan, West Bengal, India. Mail ID of Urmimala Sen: usen@microbio.buruniv.ac.in Mail ID of Subhrakanti Mukhopadhyay: skmukhopadhyay@microbio.buruniv.ac.in Haladaptatus sp strain R4 has been isolated from SreeMa saltern of Ramnagar, West Bengal, India. Salterns of this area are having wide prokaryotic diversity that can be utilized in the field of bioprospecting in pharmaceutical industry, agriculture and environmental cleaning. Strain R4 being a member of genus Haladaptatus is euryhaline in nature, capable of withstanding wide salinity range and complete absence of NaCl for many hours. Haladaptatus sp strain R4 is loaded with enzymes and proteins of therapeutic values. The strain codes for anti-cancer enzymes like L-Asparaginase and L-Glutaminase. Both the enzymes are reported to be used as chemotherapeutic agents to treat variety of lymphoproliferative disorders and lymphomas particularly Acute Lymphoblastic Leukaemia (ALL) and Hodgkin’s lymphoma. N-acyl homoserine lactone dependent quorum sensing is related to virulence in Gram-negative pathogens. Strain R4 produces two quorum quenching enzymes N-Acyl-Homoserine Lactone Lactonase and acylase that can destroy the structure of N-acyl homoserine lactone. Thus pathogenicity would be destroyed without insertion of any drug inside the pathogen, avoiding resistance complications (antibiotic resistance is an emerging problem in today’s antibiotic dependant pathogen therapy). This archaea is special in having both “salt-in-cytoplasm” and “organic osmolyte” mechanism to withstand the osmotic shock of its environment. Glycine betaine along with trehalose is compatible solutes of strain R4. Glycine betaine can stabilize cells under salt, temperature or desiccation stress. The archaea can be used as a novel “cell factory” of glycine betaine for “Bacterial Milking”. “Bacterial Milking” is a biotechnological process where prokaryotic cells can be used for production of compatible solutes by giving osmotic stress. The strain is having a retinal like pigment with strong UV-absorbing and anti-oxidant properties. Retinal is another name of miracle in skin care. It reduces sun damage, wrinkles and known for cell renewal. Production of Poly-Hydroxy Butyrate (PHB) by this strain adds to its applicability in environmental cleaning. Production of enzymes like cellulase, amylase, lipase, protease, tryptophanase and phosphate solubilisation are also observed in vitro and in silico. ANI value of Haladaptatus sp strain R4 with its closest relative Haladaptatus paucihalophilus DX253 calculated in Kostas Lab, IMG JGI and EzBioCloud is less than 95%, revealing the fact that strain R4 is a new species within the genus Haladaptatus. Strain R4 is a pink colored coccus which grows optimally at 37oC, 3M NaCl, pH 5.5 and 2% Mg++ ion concentration. It is a catalase, indole and MR (Methyl Red Test) positive and VP (Vogues-Proskauer Test) negative strain. It is positive for nitrate reduction test. The strain is capable of utilizing a number of carbohydrates starting from Dextrose, Lactose, Galactose, Maltose, Fructose, Sucrose, L-arabinose, Mannose, Sorbitol, Mannitol and many more. Strain R4 grows in presence of heavy metals like arsenic, lead, cadmium, chromium and mercury. The strain is sensitive to all the antibiotics tested. The tested antibiotics are Bacitracin, Chloramphenicol, Gentamycin, Vancomycin, Penicillin-G, Neomycin, Polymyxin-B, Rifampicin and Nalidixic acid. Pathogenicity Island, antibiotic resistance genes and coagulase gene are not coded in the whole genome of the strain. Hence, it can be concluded that this archaea is not harmful to the eukaryotes. Haladaptatus sp strain R4 could be used as a potential novel archaeal source of therapeutic agents. The whole-genome has been deposited at DDBJ/ENA/GenBank under the accession number LWHG00000000. RefSeq Identifier is GCF_001625445.1. The 16S rDNA sequence is deposited at NCBI with accession number KT174478.
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Two-thirds of reported gut microbiome-disease associations are not immediately reproducible
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Two-thirds of reported gut microbiome-disease associations are not immediately reproducible Braden T Tierney1,2,3,4, Yingxuan Tan1, Zhen Yang2,3,4, Bing Shui5, Michaela J Walker6, Benjamin M Kent7, Aleksandar D Kostic2,3,4+, Chirag J Patel1+ +co-corresponding author Lead contacts: Chirag J Patel chirag_patel@hms.harvard.edu Aleksandar D Kostic Aleksandar.Kostic@joslin.harvard.edu 1Department of Biomedical Informatics, Harvard Medical School, Boston, MA 02115, USA 2Section on Pathophysiology and Molecular Pharmacology, Joslin Diabetes Center, Boston, MA 02215, USA 3Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA 02215, USA 4Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA 5Department of Cancer Biology, Dana Farber Cancer Institute 6Independent 7US Marine Corps Reproducibly associating human gut microbes with disease is prerequisite to understanding the role of the microbiome in human health. We aimed to reproduce 581 species-level associations for 6 prevalent and well-studied diseases using 15 public cohorts (2,343 samples). We used “vibration of effects” to find robust disease indicators, reproducing only 162 associations (27.9%) and identifying a preponderance of “janus effects” -- where associations can be positive or negative depending on slight changes to modeling strategy. Published associations in type 1 and type 2 diabetes were particularly non-robust. We reproduced prior findings implicating bias, showing that features like medication (e.g. metformin) and body mass index (BMI) confound associations. Further, we highlighted the influence of other important variables like sequencing depth, glucose levels, cholesterol, and age. Overall, our results demonstrate that simple un-adjusted correlations or single modeling strategies in isolation are not sufficient to engender reproducible or biologically informative conclusions.
Speaker(s):A commensal Clostridium species protects against inflammation by modulating ILC3s Chin Yee Tan1,2, Neeraj K. Surana1,2,3 1Department of Molecular Genetics and Microbiology; 2Department of Pediatrics; 3Department of Immunology, Duke University School of Medicine The microbiome is intricately linked to human health and, when dysregulated, can cause disease. Recently, it has been shown that specific members of the intestinal microbiota regulate immunity, a finding that offers an approach for treating autoimmune disease through microbiome engineering. In previously published work, we identified Clostridium immunis, a new human-derived commensal bacterium that protects mice against colitis. To understand the host immunological response to C. immunis, we performed comprehensive flow cytometric analysis of the colonic immune system of C. immunis-treated mice, as well as cytokine analysis of mouse colonic tissue cultured ex vivo with C. immunis. Interestingly, we observed that the number of group 3 innate lymphoid cells (ILC3s), as well as ILC3-produced cytokines were significantly decreased with C. immunis treatment. ILC3s are a rare but critical tissue-resident cell population known to have protective roles in certain enteric infections, but also exert pathogenic effects in autoimmune disease. We studied if C. immunis is able to modulate the outcomes of diseases known to be attenuated or exacerbated by ILC3s—Citrobacter rodentium infection and a murine model of multiple sclerosis (MS), respectively. Accordingly, C. immunis administration led to worse disease in Citrobacter rodentium infection, but conversely was protective in the MS model. Lastly, we investigated if C. immunis was also able to modulate the human immune system. Indeed, co-culturing colonic biopsies from patients with inflammatory bowel disease with C. immunis led to a decrease in inflammatory cytokines. Considered together, we have identified a commensal intestinal bacterium that exerts a potent immunomodulatory effect on inflammatory disease, likely through its effect on ILC3s.
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Temporally evaluating the Anal Cancer Microbiome during Chemoradiation
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Temporally evaluating the Anal Cancer Microbiome during Chemoradiation Daniel Lin, Ramez Kouzy, Molly El Alam, Joseph Abi Jaoude, Bruce Minksy, Emma Holliday, Ann Klopp, Prajnan Das, Cathy Eng, Van Morris, Lauren Colbert, and Cullen Taniguchi *The University of Texas MD Anderson Cancer Center, Houston, TX, United States Objective: Despite high rates of 5-year survival for localized HPV-driven anal squamous cell carcinoma (ASCC), approximately 20% of patients succumb to disease after chemoradiation therapy (CRT). Microbiota play a significant role in the pathogenesis and protection of cervical and oropharyngeal cancers, but its role in ASCC remains unknown. The goal of our ongoing clinical trial is to characterize the local microbiome of ASCC and its changes throughout radiation therapy. Methods: We are currently enrolling patients with localized HPV-related ASCC undergoing external beam radiation treatment with concurrent 5-FU and cisplatin. Serial anorectal microbiota samples were collected utilizing a non-invasive swab biopsy technique at baseline, week 1, 3, and 5 (end of treatment) during CRT and at 3 months post-CRT. Microbiome sequencing was performed using 16s rRNA sequencing. Shannon Diversity Index (SDI) was used to measure alpha diversity. PERMANOVA was used for multivariate comparisons. Linear regression was used to evaluate temporal changes. Linear Discriminant Effect Size (LEFSe) analysis was used to identify changes in relative abundance of specific taxa. Results: This analysis includes 22 patients. Diversity of the anorectal microbiome decreased throughout chemoradiation treatment with Week 5 samples exhibiting decreased diversity vs. baseline (SDI: P = 0.028). A subsequent increase in diversity was observed for 3 months follow-up (SDI: P = 0.033). Multivariate analysis demonstrated unique bacteria populations for baseline vs. end of treatment (P = 0.027). Linear regression revealed increased Corynebacteriales (R2 = 0.20, P = 0.0002) and decreased Clostridia (R2 = 0.12, P = 0.003) throughout CRT. LEFse analysis revealed an enrichment of Corynebacteriales and a depletion of Clostridia at end of treatment compared to both baseline and 3 months follow-up. Conclusion: Chemoradiation of the ASCC results in significant shifts in overall diversity, composition, and specific taxa throughout treatment. However, many of these changes appear to resolve 3 months post-CRT, with follow-up samples exhibiting microbial profiles similar to baseline. Further investigation into the effects of radiation therapy on the tumor microbiome may lead to unique approaches in improving outcomes for ASCC patients.
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Gut Dysbiosis in Patients with Pulmonary Arterial Hypertension
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Gut Dysbiosis in Patients with Pulmonary Arterial Hypertension Daphne Moutsoglou1, Matthew Hamilton2, E. Kenneth Weir3, Alexander Khoruts1, Thenappan Thenappan3 1. Division of Gastroenterology, Hepatology, and Nutrition. Department of Medicine. University of Minnesota, Minneapolis. 2. Cardiovascular Division, Department of Medicine. University of Minnesota, Minneapolis 3. Biotechnology Institute. College of Biological Sciences, University of Minnesota, Minneapolis. Background: The pathophysiology of pulmonary arterial hypertension (PAH) is poorly understood but may occur from remodeling of the extracellular matrix with fibrosis of pulmonary vessels that is primarily driven by perivascular inflammation and immune dysregulation. Recent studies suggest a role for the gut-pulmonary axis in pulmonary and cardiovascular disease. Hypothesis: We hypothesized that gut dysbiosis and barrier dysfunction lead to an altered burden of circulating microbial metabolic products and bacterial components, resulting in perivascular inflammation that promotes PAH. Methods: DNA was isolated from the fecal samples of 35 PAH patients and 36 healthy controls, and the V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using the Illumina platform. Sequences were analyzed using mothur software (open source, www.mothur.org) and Linear discriminant analysis Effect Size (LEfSe). Results: On principal component analysis, patients with PAH had a distinct intestinal microbiome composition compared to healthy controls (Figure 1A). Additionally, the Shannon diversity index was significantly lower in PAH patients (3.394 ± 0.08 vs. 3.938 ± 0.04, p
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Immuno-Reactive Cancer Organoid Models to Examine Microbiome Metabolite Effects on Immune Checkpoint Blockade Efficacy
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Immuno-Reactive Cancer Organoid Models to Examine Microbiome Metabolite Effects on Immune Checkpoint Blockade Efficacy Ethan Shelkey1, David Soto-Pantoja2, Yong Lu3, Shay Soker1,4 1Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC; 2Hypertension and Vascular Research Center, Wake Forest School of Medicine, Winston-Salem, NC; 3Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC; 4Comprehensive Cancer Center at Wake Forest Baptist Medical, Winston-Salem, NC Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, a sizable percentage of patients are non-responders to all types of immunotherapy. Yet, there has been limited 3D in-vitro models to assess tumor immune-reactivity. These systems are ideal for isolating mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic responses in patients. Methods: We created organoids consisting of tumor and immune cells, embedded in extracellular matrix-like hydrogels. Organoids were treated with therapeutic equivalent doses of αPD-1 and αCTLA-4 or single dose of αCD-47 and viability assays, flow cytometry, RT-qPCR, and IHC staining were performed to determine the effects of ICB. Physiologic concentrations of metabolites derived from likely effector bacterial species were then evaluated for their effects on genetic expression and immunotherapy efficacy. Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR resulted in some elucidation of potential cellular changes due to bacterial metabolites with further quantification ongoing. Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We can now continue to change individual microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive to ICB treatment.
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Pro-longevity bacterial colanic acid regulates host mitochondrial dynamics through the endo-lysosomal system and transcriptional response
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Pro-longevity bacterial colanic acid regulates host mitochondrial dynamics through the endo-lysosomal system and transcriptional response Guo Hu1,2, Mooncheol Park1, and Meng Wang1,2 1Huffington Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA; 2Department of Genetics and Genomics, Baylor College of Medicine, Houston TX 77030, USA. Irreversible and inevitable, aging occurs in every organism. Confronting soaring number of age-related diseases, to promote healthy aging is more urgent than ever. The gut microbiota effectively impacts the host’s physiological functions and the progression of aging. To detangle the complex network of host-microbiota interaction, we took advantages of the genetic tractability and the short lifespan of Caenorhabditis elegans to dissect the effect of microbial genetics on host longevity. Through systemically screening the Escherichia coli deletion library, our previous findings revealed that host C. elegans live longer when raised on the Lon protease deletion mutant of E. coli (∆lon) that overproduces exopolysaccharide – Colanic Acid (CA). We further discovered that CA is sufficient to promote longevity, and both the ∆lon and CA induce mitochondrial fission in the host. To understand how bacterial CA signals to host mitochondria, we hypothesized endo-lysosomal system facilitates CA uptake to host intestinal cells. We found that genetic knock-down of key regulators in the endocytic pathway fully suppressed the CA-induced mitochondrial fission and lifespan extension. Through profiling lysosomal proteomics, we found that CA increases the interaction of lysosomes with mitochondria. Furthermore, through transcriptome analysis, we identified several candidates of the hsf-1 transcription factor are up-regulated in the host raised on the ∆lon. RNAi inactivation of hsf-1 fully suppresses the lifespan extension and mitochondrial fission in the host caused by ∆lon. Together, our results suggest that the host’s endo-lysosome system may facilitate CA transportation into host’s intestinal cells and mediate the interaction with mitochondria, which consequently induces specific transcriptional responses to promote mitochondrial homeostasis and organism longevity. On-going studies will examine the targets of hsf-1 and the role of lipid metabolism in the CA-mediated longevity mechanism. Understanding the longevity mechanisms of bacterial CA will provide new strategies in probiotic development to combat with age-related diseases.
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Gut microbial proinflammatory proteolytic activity is detected before ulcerative colitis onset
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Gut microbial proinflammatory proteolytic activity is detected before ulcerative colitis onset Heather J Galipeau1, Alberto Caminero1, Williams Turpin2,3, Miriam Bermudez-Brito1, Alba Santiago1, Josie Libertucci1, Marco Constante1, Juan Antonio Raygoza Garay2,3, Gaston Rueda1, Sarah Armstrong1, Alex Clarizio1, Michelle I. Smith2, Michael G. Surette1, Premysl Bercik1, The CCC GEM Project Research Consortium, Kenneth Croitoru2,3, Elena F. Verdu1 1. Farncombe Family Digestive Health Research Institute, McMaster University, Hamilton, Canada. 2. Zane Cohen Centre for Digestive Diseases, Lunenfeld Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada. 3. Division of Gastroenterology, Department of Medicine, University of Toronto, Toronto, Ontario, Canada Introduction: Altered gut microbiota composition and function has been associated with inflammatory bowel diseases (IBD) including ulcerative colitis (UC), but causality and mechanisms remain unknown. Most studies have examined patients with active or treated disease, making it difficult to discern if the described changes are attributable to the effect of inflammation. Little is known about microbial compositional or functional changes that occur before disease onset. Aims: We studied a longitudinal cohort of subjects at risk for IBD to define the fecal microbial composition and function in subjects prior to UC onset (pre-UC) and at diagnosis (post-UC), and in matched at-risk subjects that remained healthy. Methods: Fecal samples were collected from healthy individuals at-risk for IBD (pre-UC; n=13) and subjects were followed longitudinally until UC diagnosis (post-UC; n=9), at which point another fecal sample was collected. Fecal samples from a cohort of matched at-risk individuals that did not develop UC were used as healthy controls (HC; n=48). We applied 16S rRNA gene sequencing, next generation shotgun sequencing, in vitro proteolytic assays and gnotobiotic colonizations to define the microbial fecal composition and proteolytic function. Results: Microbiota composition of post-UC subjects was different from HC and pre-UC. Post-UC subjects clustered separately from pre-UC and HC, based on bray-curtis and unweighted UniFrac, had reduced alpha-diversity compared to pre-UC and HC and had reduced abundance of Adlercreutzia compared to pre-UC and HC. In vitro functional analysis revealed increased fecal proteolytic and elastase activity in pre-UC and post-UC samples, compared to HC. Metagenomics identified pathways and gene families related to protein metabolism and proteases/peptides that were significantly different between HC and pre-UC samples, suggesting a bacterial component to the pre-UC proteolytic signature. Elastase activity inversely correlated with the relative abundance of Adlercreutzia, and other potentially beneficial taxa, and directly correlated with Bacteroides vulgatus, a known proteolytic taxon. High elastase activity was confirmed in Bacteroides isolates from fecal samples. Bacterial contribution and functional significance of the proteolytic signature was investigated in germ-free adults and litters born from dams colonized with HC, pre-UC or post-UC microbiota. Mice colonized as adults, or neonatally with pre-UC microbiota developed higher fecal proteolytic activity and an inflammatory immune tone compared with HC colonized mice. Conclusion: We have identified increased fecal proteolytic activity that precedes clinical diagnosis of UC and associates with gut microbiota changes. This may constitute a non-invasive biomarker of inflammation to monitor at-risk populations that can be targeted therapeutically with anti-proteases.
Speaker(s):Adequacy of calcium and vitamin D enriches probiotic bacteria and reduces dysbiotic Parasutterela bacteria and inflammation in the colon of C57BL/6 mice fed a Western-style diet Huawei Zeng1*, Bryan D Safratowich1, Zhenhua Liu2, Michael R Bukowski1, Suzanne L Ishaq3 1U. S. Department of Agriculture, ARS, Grand Forks Human Nutrition Research Center, Grand Forks, ND 58203; 2 School of Public Health, Univ of Massachusetts, Amherst, MA 01003; 3 School of Food and Agriculture, Univ of Maine, Orono, ME 04469 * Correspondence to: huawei.zeng@usda.gov Adoption of an obesogenic diet low in calcium and vitamin D (CaD) leads to increased obesity, colonic inflammation, and cancer. However, the mechanism remains to be elucidated. We tested the hypothesis that CaD supplementation may reduce colonic inflammation, oncogenic signaling, and dysbiosis in the colon of C57BL/6 mice fed a Western diet. Male C57/BL6 mice (4-wk old) were assigned to 3 dietary groups for 36 wks: (1) AIN76A as a control diet (AIN); (2) a defined rodent “new Western diet” (NWD); or (3) NWD with CaD supplementation (NWD/CaD). Compared to the AIN, mice receiving the NWD or NWD/CaD exhibited more than 0.2-fold increase in the levels of plasma leptin, tumor necrosis factor α (TNF-α) and body weight. The levels of plasma interleukin 6 (IL-6), inflammatory cell infiltration, and b-catenin/Ki67 protein (oncogenic signaling) were increased more than 0.8-fold in the NWD (but not NWD/CaD) group compared to the AIN group. Consistent with the inflammatory phenotype, colonic secondary bile acid (BA, inflammatory bacterial metabolite) levels increased more than 0.4-fold in the NWD group compared to the NWD/CaD and AIN groups. Furthermore, the α diversity of colonic bacterial species, indicative of health, was decreased by 30%; and the abundance of colonic Proteobacteria phylum (e.g., Parasutterela genus), considered signatures of dysbiosis, was increased more than 4-fold in the NWD group compared to the AIN and NWD/CaD groups. However, only the abundance of Parasutterella was positively correlated with inflammatory cell infiltration. These data indicate that Parasutterella genus may be a more sensitive dysbiosis marker than Proteobacteria phylum in the context of Western diet consumption. Collectively, CaD adequacy enriches probiotic bacteria and reduces dysbiotic bacteria and inflammation in the colon of mice fed a Western diet. Acknowledgement: This work was funded by USDA, ARS, CRIS project (3062-51000-050-00D).
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