(172f) Gut Microbiota-Derived Tryptophan Metabolites Modulate Inflammatory Response in Hepatocytes and Macrophages

Gut
Microbiota-derived Tryptophan Metabolites Modulate Inflammatory Response in
Hepatocytes and Macrophages

Smitha
Krishnan¹, Yufang Ding², Maria Choi¹, Nima
Saedi³, Martin L. Yarmush³, Arul Jayaraman²,
and Kyongbum Lee¹

1.
Department of Chemical and Biological Engineering, Tufts University, Medford,
MA 02155

2.
Department of Biomedical Engineering, Texas A&M University, College
Station, TX 77843

3.
Center for Engineering in Medicine, Massachusetts General Hospital, Boston, MA
02114

The human gastrointestinal (GI) tract harbors an
estimated 10 to 100 trillion microorganisms, encompassing several hundred
different species. Collectively referred to as the gut microbiota, the microbes
perform a number of essential physiological functions, including the metabolism
of complex carbohydrates [1], development of the immune system [2], and defense
against pathogens [3]. Beyond the GI tract, gut-brain, gut-lung, and gut-liver
associations have also been identified, highlighting the importance of the
microbiota in host physiology. Alterations in the intestinal microbiota
composition (i.e. dysbiosis) have been correlated to various diseases,
including obesity [4, 5], diabetes [6], and cancer [7].

Non-alcoholic
fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver
disease in Western countries. It is characterized by steatosis, or fat
deposition in the liver, which can remain a benign condition. However, in
10-20% of patients, NAFLD progresses from steatosis to non-alcoholic
steatohepatitis (NASH), which can lead to cirrhosis and liver cancer. Direct,
potentially causal involvement of the microbiota in NASH was highlighted by Le
Roy et al. [11], who reported that high fat diet (HFD)-fed germ-free (GF) mice
inoculated with commensal bacteria from GI tract of HFD-fed hyperglycemic mice
developed steatohepatitis, whereas HFD-fed GF mice inoculated with bacteria
from HFD-fed, but normoglycemic mice only showed mild steatosis. This suggests
that the composition of the intestinal microbiota, and hence their metabolic
products, could determine whether NAFLD progresses to NASH. The study described
in this abstract utilizes metabolomics in conjunction with biochemical assays to
identify and characterize microbiota-dependent immunomodulatory metabolites
that could modulate liver inflammation in steatosis.

            In previous
work [13], we identified a panel of aromatic amino acid (AAA)-derived
metabolites whose levels in the mouse intestine depended on the presence of the
microbiota. Furthermore, several of these metabolites were found to activate
the aryl hydrocarbon receptor (AhR), a transcriptional regulator of
host-microbiota interactions in the intestine. We compared the profiles of these
metabolites in serum, cecum, and liver samples from 14-week old male C57BL/6J
mice raised for 8 weeks either on HFD or low-fat diet (LFD). Untargeted LC-MS
experiments found that several of the previously identified gut
microbiota-dependent metabolites were significantly depleted in HFD mice
compared to LFD mice (Figure 1a-c), including Ahr activators tryptamine (TA)
and indole-3-acetate (I3A) (Figure 1d).

            We
next investigated the effects of TA and I3A on inflammatory responses in
hepatocytes using both human (HepG2) and murine (AML12) cell lines. Cultured
HepG2 and AML12 cells were treated with 10, 100, or 500 uM TA or I3A for 24 h,
and then exposed to the pro-inflammatory cytokine TNF_ for 24 h in the presence
of the metabolites. In both human and murine-derived hepatocytes, pretreatment
with TA or I3A significantly attenuated TNF_-stimulated increases in
intracellular fatty acid levels, with I3A exhibiting greater potency.
Similarly, pretreatment with TA or I3A significantly attenuated TNF_Ðstimulated
increases in intracellular bile acid levels. These effects were also observed
when the hepatocytes were preloaded with lipid droplets prior to metabolite and
cytokine treatment by growing the cells in fatty acid supplemented culture
medium to mimic a steatotic state.

To confirm that
the metabolites can activate the AhR pathway in hepatocytes, we applied TA and
I3A to H1G1.1c3 cells expressing a stable enhanced green fluorescent protein
(EGFP) reporter construct regulated by the AhR response element (AHRE) derived
from the CYP1A1 promoter [14]. Consistent with our previous findings [13], 24 h
exposure to TA or I3A dose-dependently increased GFP expression in the reporter
cells, indicating ligand activation of the AhR in hepatocytes by these
metabolites.

Finally, we investigated
the effects of I3A and TA on macrophages, as polarization of resident
macrophages towards a pro-inflammatory state is a key step in the progression
of steatohepatitis. RAW 264.7 murine macrophages were treated with I3A and TA
for 4 h, and then stimulated with palmitate for 18 h followed by LPS exposure for
6 h in the presence of the metabolites. Treatment with I3A significantly
attenuated the induction of the pro-inflammatory cytokines TNFa, IL-1b and MCP-1 by
palmitate and LPS in a dose dependent manner at both mRNA and protein levels. Unlike
I3A, TA only decreased the gene expression of IL-1b. However, TA
significantly decreased TNFa and MCP-1 expression at the protein
level. Both I3A and TA significantly decreased bone marrow-derived macrophage (BMDM)
migration towards MCP-1 in a dose dependent manner.           

In summary, our
results show that several microbiota-dependent metabolites derived from
tryptophan are significantly depleted in a murine model of diet-induced liver
steatosis, and that these metabolites can act directly on hepatocytes and
macrophages to modulate inflammatory pathways. Our results demonstrate that
these microbiota metabolites are ligands for the AhR. Taken together, our
findings support the hypothesis that dysbiosis of the gut microbiota could
predispose the liver to inflammation in diet-induced steatosis through an
altered microbiota metabolite profile. Prospectively, insights into the
mechanisms underlying the link between microbiota dysbiosis and NAFLD could
provide novel strategies to treat or prevent the progression of fatty liver
diseases through the use of probiotics or postbiotics.

Figure 1

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