Endotoxin Producers Overgrowing in Human Gut Microbiota as the Causative Agents for Nonalcoholic Fatty Liver Disease - PubMed (original) (raw)

Endotoxin Producers Overgrowing in Human Gut Microbiota as the Causative Agents for Nonalcoholic Fatty Liver Disease

Na Fei et al. mBio. 2020.

Abstract

Gut microbiota-derived endotoxin has been linked to human nonalcoholic fatty liver disease (NAFLD), but the specific causative agents and their molecular mechanisms remain elusive. In this study, we investigated whether bacterial strains of endotoxin-producing pathogenic species overgrowing in obese human gut can work as causative agents for NAFLD. We further assessed the role of lipopolysaccharide (LPS)-Toll-like receptor 4 (TLR4) cross talk in this pathogenicity. Nonvirulent strains of Gram-negative pathobionts were isolated from obese human gut and monoassociated with C57BL/6J germfree (GF) mice fed a high-fat diet (HFD). Deletion of waaG in the bacterial endotoxin synthetic pathway and knockout of TLR4 in GF mice were used to further study the underlying mechanism for a causal relationship between these strains and the development of NAFLD. Three endotoxin-producing strains, Enterobacter cloacae B29, Escherichia coli PY102, and Klebsiella pneumoniae A7, overgrowing in the gut of morbidly obese volunteers with severe fatty liver, induced NAFLD when monoassociated with GF mice on HFD, while HFD alone did not induce the disease in GF mice. The commensal Bacteroides thetaiotaomicron (ATCC 29148), whose endotoxin activity was markedly lower than that of Enterobacteriaceae strains, did not induce NAFLD in GF mice. B29 lost its proinflammatory properties and NAFLD-inducing capacity upon deletion of the waaG gene. Moreover, E. cloacae B29 did not induce NAFLD in TLR4-deficient GF mice. These nonvirulent endotoxin-producing strains in pathobiont species overgrowing in human gut may work as causative agents, with LPS-TLR4 cross talk as the most upstream and essential molecular event for NAFLD.IMPORTANCE Recent studies have reported a link between gut microbiota and nonalcoholic fatty liver disease (NAFLD), showing that germfree (GF) mice do not develop metabolic syndromes, including NAFLD. However, the specific bacterial species causing NAFLD, as well as their molecular cross talk with the host for driving liver disease, remain elusive. Here, we found that nonvirulent endotoxin-producing strains of pathogenic species overgrowing in obese human gut can act as causative agents for induction of NAFLD and related metabolic disorders. The cross talk between endotoxin from these specific producers and the host's TLR4 receptor is the most upstream and essential molecular event for inducing all phenotypes in NAFLD and related metabolic disorders. These nonvirulent endotoxin-producing strains of gut pathogenic species overgrowing in human gut may collectively become a predictive biomarker or serve as a novel therapeutic target for NAFLD and related metabolic disorders.

Keywords: fatty liver; gut inflammation; intestinal microbiology; nonalcoholic steatohepatitis.

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Figures

FIG 1

E. cloacae B29-dependent induction of NAFLD in GF mice under HFD feeding requires endotoxin expression. Germfree (GF) mice were fed either normal chow diet (NCD) or high-fat diet (HFD) and inoculated with either Luria-Bertani (LB), wild-type E. cloacae B29, or the endotoxin-lacking waaG mutant strain (data were collected at the end of 15 weeks after inoculation). (A) Liver histology (hematoxylin and eosin stain). Scale bar, 50 μm. (B) NAFLD activity score. (C) Liver triglyceride. (D) Body weight. (E) Mass of epididymal, mesenteric, subcutaneous inguinal, and retroperitoneal fat pads. (F) Adipocyte mean area. (G) Oral glucose tolerance test (OGTT) and the area under the curve (AUC) for the plasma glucose concentration (P < 0.05; &, HFD+B29 versus other groups; #, HFD+LB and HFD+B29-mutant versus NCD groups). (H) Plasma insulin concentration before (fasting) and 30 min after oral glucose load. Data shown are means ± SEM (n = 5 to 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 2

E. cloacae B29 induction of systemic and local inflammation in HFD-fed NAFLD GF mice is dependent upon endotoxin expression. GF mice were fed either normal chow diet (NCD) or high-fat diet (HFD) and inoculated with either Luria-Bertani broth (LB), wild-type E. cloacae B29, or the endotoxin-lacking waaG mutant strain (data were collected at the end of 15 weeks after inoculation). (A) ELISA of serum LPS-binding protein (LBP). (B) ELISA of serum amyloid A (SAA-3). (C) RT-qPCR analysis of expression of the Tlr4 gene in the liver. (D to F) RT-qPCR analysis of expression of the Tnfα, Il1β, Mcp1, Ikkε, and Reg3γ genes in the liver (D), ileum (E), and epididymal fat pad (F). All mRNA quantification data were normalized against the housekeeping gene. Gene expression levels were expressed as values relative to those of the control group (NCD+LB). Tlr4, toll-like receptor 4 gene; Tnfα, tumor necrosis factor-α gene; Il1β, interleukin-1β gene; Mcp1, monocyte chemoattractant protein-1 gene; Ikkε, I kappa B kinase epsilon gene; Reg3γ, c-type lectin regenerating islet-derived protein 3-γ. Data shown are means ± SEM (n = 5 to 10). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 3

Absence of TLR4 in mice prevented NAFLD induced by the LPS-producing gut opportunistic pathobiont Enterobacter cloacae B29 (data were collected at the end of 15 weeks after inoculation). (A) Liver histology (hematoxylin and eosin stain) of TLR4 mutant (TLR4−/−) mice with or without B29 at the end of the trial. Scale bar, 100 μm. (B) NAFLD activity score. (C) Body weight. (D) Mass of epididymal, mesenteric, subcutaneous inguinal, and retroperitoneal fat pads of TLR4−/−mice with or without B29. (E) Plasma insulin concentration before (fasting) and 30 min after oral glucose load in TLR4−/− mice with or without B29. (F) ELISA of serum leptin in TLR4−/− mice with or without B29 (after adjustment for body weight). (G) ELISA of serum LBP in TLR4−/− mice with or without B29. (H) ELISA of serum amyloid A (SAA-3) in TLR4−/− mice with or without B29. Data shown are means ± SEM (n = 5 to 9). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG 4

Gram-negative bacteria producing LPS with different endotoxin activity levels showed different capacities to induce NAFLD in HFD-fed GF mice (data were collected at the end of 15 weeks after inoculation). (A) Liver histology (hematoxylin and eosin stain). Scale bar, 50 μm. (B) NAFLD activity score. (C) Liver triglyceride. (D) Body weight. (E) Mass of epididymal, mesenteric, subcutaneous inguinal, and retroperitoneal fat pads. (F) Adipocyte mean area. (G) Oral glucose tolerance test (OGTT) and the area under the curve (AUC) for the plasma glucose concentration (P < 0.05; #, HFD+LB versus HFD+B.theta; &, HFD+LB versus HFD+Kleb; *, HFD+LB versus HFD+E.coli). (H) Plasma insulin concentration before (fasting) and 30 min after oral glucose load. Data shown are means ± SEM (n = 7 to 16). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

References

1. Loomba R, Sanyal AJ. 2013. The global NAFLD epidemic. Nat Rev Gastroenterol Hepatol 10:686–690. doi: 10.1038/nrgastro.2013.171. - DOI - PubMed
1. Tripathi A, Debelius J, Brenner DA, Karin M, Loomba R, Schnabl B, Knight R. 2018. The gut–liver axis and the intersection with the microbiome. Nat Rev Gastroenterol Hepatol 15:397–411. doi: 10.1038/s41575-018-0031-8. - DOI - PMC - PubMed
1. Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, Semenkovich CF, Gordon JI. 2004. The gut microbiota as an environmental factor that regulates fat storage. Proc Natl Acad Sci U S A 101:15718–15723. doi: 10.1073/pnas.0407076101. - DOI - PMC - PubMed
1. Rabot S, Membrez M, Bruneau A, Gerard P, Harach T, Moser M, Raymond F, Mansourian R, Chou CJ. 2010. Germ-free C57BL/6J mice are resistant to high-fat-diet-induced insulin resistance and have altered cholesterol metabolism. FASEB J 24:4948–4959. doi: 10.1096/fj.10-164921. - DOI - PubMed
1. Le Roy T, Llopis M, Lepage P, Bruneau A, Rabot S, Bevilacqua C, Martin P, Philippe C, Walker F, Bado A, Perlemuter G, Cassard-Doulcier A-M, Gérard P. 2013. Intestinal microbiota determines development of non-alcoholic fatty liver disease in mice. Gut 62:1787–1794. doi: 10.1136/gutjnl-2012-303816. - DOI - PubMed

Endotoxin Producers Overgrowing in Human Gut Microbiota as the Causative Agents for Nonalcoholic Fatty Liver Disease - PubMed (original) (raw)