Using Metabolomics to Identify Gut Microbiota-Derived Metabolites As Host Inflammation Modulators

Lee, K., Tufts University
Krishnan, S., Tufts University
Choi, M., Tufts University
Ding, Y., Texas A&M University
Saedi, N., Massachusetts General Hospital
Jayaraman, A., Texas A&M University
Yarmush, M. L., Harvard Medical School

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease in Western countries, paralleling the rise in obesity. It is characterized by steatosis (fat deposition in the liver), which can remain a benign condition. However, in ~20% of patients, NAFLD progresses from steatosis to steatohepatitis, which can lead to cirrhosis and liver cancer. Despite accumulating evidence that the gut microbiota plays a mechanistic role, the molecular mediators and cellular pathways involved remain unclear. Recent findings, including our own work [1], demonstrated that microbiota species produce bioactive metabolites that engage host cellular pathways. The present study investigates the hypothesis that gut microbiota dysbiosis perturbs the balance of immunomodulatory microbiota metabolites, exacerbating liver inflammation in steatosis.

We compared the metabolite profiles in the cecum of germ-free (GF) and conventionally raised (CONV-R) mice, and identified metabolites whose levels in the mouse intestine depend on the presence of the microbiota. Focusing on metabolites depleted in GF mice, we found derivatives of aromatic amino acids (AAAs), several of which we had previously [1] shown to activate the aryl hydrocarbon receptor (AHR), a transcriptional regulator of host-microbiota interactions. To investigate a potential role for these metabolites in NAFLD, we compared their profiles in serum and liver samples from 14-week old male C57BL/6J mice raised for 8 weeks either on high-fat diet (HFD) and low-fat diet (LFD) mice. Untargeted metabolomics detected microbiota-dependent AAA derivatives in serum and liver samples from both HFD and LFD mice. We selected a pair of representative metabolites from this set, tryptamine (TA) and indole-3-acetate (I3A), for further analysis using in vitro culture models. Cultured HepG2 and AML12 cells were treated with 10-500 mM TA or I3A for 24 h, and then exposed to the pro-inflammatory cytokine TNFα for another 24 h in the presence of the metabolites. We focused on bile and fatty acids as functional readouts, as dysregulation of lipid metabolism is a hallmark of NAFLD. Compared to vehicle control, TNFα treatment significantly increased both intra- and extracellular levels of several bile acids. Pretreatment with either TA or I3A significantly attenuated the increases in bile acid levels. Similarly, TNFα treatment significantly increased both intra- and extracellular levels of several free fatty acids, notably palmitate. Pretreatment with I3A, but not TA, significantly attenuated these increases. To further delineate the signaling and regulatory pathways affected by the metabolites, we performed an untargeted proteomics analysis. Cytokine treatment significantly decreased the level of albumin, while increasing the expression of several acute phase proteins. Pretreatment with I3A, but not TA, attenuated this trend.

In summary, we show that the levels of AAA-derived microbiota metabolites are significantly depleted in a diet model of liver steatosis, and that these metabolites can act directly on hepatocytes, possibly through the AHR, to modulate inflammatory pathways. Ongoing work utilizes metabolomics and proteomics to investigate potential crosstalk between AHR and other pathways regulating liver lipid metabolism. Insights into the mechanisms linking dysbiosis and NAFLD could provide novel strategies to treat fatty liver diseases through pre-/pro-/postbiotics.