Summary of Study ST004229

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002668. The data can be accessed directly via it's Project DOI: 10.21228/M8SG2M This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST004229
Study TitleAromatic Microbial Metabolite Hippuric Acid Potentiates Pro-Inflammatory Responses in Macrophages through TLR-MyD88 Signaling and Lipid Remodeling - Lipidomics analysis on bone marrow derived macrophages pre-treated with hippuric acid and stimulated with M1-like (LPS+IFNγ)
Study SummaryAuthors have assessed whether hippuric acid broadly perturbs lipid metabolism by performing lipidomics analysis on bone marrow derived macrophages (BMDMs) pre-treated with hippuric acid and stimulated with M1-like (LPS+IFNγ) stimuli. Lipids were extracted 24 hours after stimulation and analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). Consistent with prior studies, M1-like macrophages displayed significant alterations in lipid composition compared to controls, including the accumulation of distinct triglyceride (TG) species and reductions in cholesterol ester (ChE) and lysophosphatidylcholine (LPC) species. Addition of hippuric acid drove changes in lipid composition in M1-polarized macrophages, with significant increases in phospholipids and neutral lipids. Lipid species such as PC(16:0_20:2), PS(19:1_18:0), and PI(18:0_17:0) increased >27-fold, and LPE(22:1), TG(18:0_22:4_22:5), and LPC(22:4) increased >4-fold. Only a few lipid species, such as PC(16:1e_17:0) and PE(16:0_14:0), were downregulated with <3-fold changes. Analysis of lipid classes revealed significant increases in phosphatidic acid, lysosphingomyelin, lysophosphatidylcholine, cholesterol ester, ceramide, and phosphatidylinositol, while phosphatidylglycerol levels decreased. This study showed that hippuric acid drove cholesterol biosynthesis and lipid remodeling in macrophages.
Institute
The Wistar Institute
Last NameShinde
First NameRahul
Address3601 Spruce Street, Philadelphia, PA, 19104, USA
Emailrshinde@wistar.org
Phone215-898-3717
Submit Date2025-09-18
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2025-10-31
Release Version1
Rahul Shinde Rahul Shinde
https://dx.doi.org/10.21228/M8SG2M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002668
Project DOI:doi: 10.21228/M8SG2M
Project Title:Aromatic Microbial Metabolite Hippuric Acid Potentiates Pro-Inflammatory Responses in Macrophages through TLR-MyD88 Signaling and Lipid Remodeling
Project Summary:The gut microbiome generates a diverse array of metabolites that actively shape host immunity, yet the pro-inflammatory potential of microbial metabolites remains incompletely understood. Using a non-targeted, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics, we identified hippuric acid, an aromatic gut microbe-derived metabolite, as a potent enhancer of pro-inflammatory responses in Escherichia coli infection model. Intraperitoneal administration of hippuric acid significantly heightened pro-inflammatory responses, promoted innate immune cell activation, and reduced survival in infected mice. Similar pro-inflammatory effects were observed in an LPS-induced inflammation model. In vitro, hippuric acid selectively potentiated M1-like macrophage polarization (LPS + IFNγ) but had no effect on M2-like polarization (IL-4). Hippuric acid further augmented responses to multiple myeloid differentiation primary response 88 (MyD88)-dependent toll-like receptor (TLR) ligands, but not to TRIF-dependent TLR3, or to cytosolic innate immune stimuli such as STING and NOD2 agonists, implicating TLR-MyD88 signaling as a likely mechanism of action. Genetic deletion of MyD88 abrogated the pro-inflammatory effects of hippuric acid both in vitro and in vivo, confirming its dependence on the MyD88 pathway. Transcriptomic and lipidomic analyses revealed that hippuric acid upregulated cholesterol biosynthesis and induced lipid accumulation. Pharmacological reduction of cellular cholesterol using fluvastatin or 25-hydroxycholesterol attenuated its pro-inflammatory effects. Notably, hippuric acid also enhanced pro-inflammatory responses in human macrophages, and its elevated levels correlated with increased sepsis mortality, underscoring its clinical relevance. Together, these findings identify hippuric acid as a previously unrecognized microbial-derived pro-inflammatory modulator that links gut microbial metabolism, lipid remodeling, and innate immune signaling, and offer new insights into its role in infection and inflammation.
Institute:The Wistar Institute
Last Name:Shinde
First Name:Rahul
Address:3601 Spruce Street, Philadelphia, PA, 19104, USA
Email:rshinde@wistar.org
Phone:215-898-3717

Subject:

Subject ID:SU004381
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Sample source
SA486100Control-2-NegControl BMDM
SA486101Control-4-PosControl BMDM
SA486102Control-4-NegControl BMDM
SA486103Control-3-PosControl BMDM
SA486104Control-3-NegControl BMDM
SA486105Control-2-PosControl BMDM
SA486106Control-1-PosControl BMDM
SA486107Control-1-NegControl BMDM
SA486108HA-LPS-IFNg-3-NegHA-LPS-IFN BMDM
SA486109HA-LPS-IFNg-3-PosHA-LPS-IFN BMDM
SA486110HA-LPS-IFNg-4-NegHA-LPS-IFN BMDM
SA486111HA-LPS-IFNg-4-PosHA-LPS-IFN BMDM
SA486112HA-LPS-IFNg-2-PosHA-LPS-IFN BMDM
SA486113HA-LPS-IFNg-2-NegHA-LPS-IFN BMDM
SA486114HA-LPS-IFNg-1-PosHA-LPS-IFN BMDM
SA486115HA-LPS-IFNg-1-NegHA-LPS-IFN BMDM
SA486116HA-4-NegHA BMDM
SA486117HA-4-PosHA BMDM
SA486118HA-3-PosHA BMDM
SA486119HA-2-PosHA BMDM
SA486120HA-2-NegHA BMDM
SA486121HA-1-PosHA BMDM
SA486122HA-1-NegHA BMDM
SA486123HA-3-NegHA BMDM
SA486124LPS-IFNg-3-NegLPS-IFN BMDM
SA486125LPS-IFNg-4-PosLPS-IFN BMDM
SA486126LPS-IFNg-4-NegLPS-IFN BMDM
SA486127LPS-IFNg-3-PosLPS-IFN BMDM
SA486128LPS-IFNg-1-NegLPS-IFN BMDM
SA486129LPS-IFNg-1-PosLPS-IFN BMDM
SA486130LPS-IFNg-2-NegLPS-IFN BMDM
SA486131LPS-IFNg-2-PosLPS-IFN BMDM
SA486132QC-1-Negn/a BMDM
SA486133QC-2-Posn/a BMDM
SA486134QC-2-Negn/a BMDM
SA486135QC-3-Posn/a BMDM
SA486136QC-3-Negn/a BMDM
SA486137QC-1-Posn/a BMDM
Showing results 1 to 38 of 38

Collection:

Collection ID:CO004374
Collection Summary:Spleens were minced with scissors and using the plunger/piston end of the syringe and tissue suspensions were passed through 100μm cell strainers. Bone marrow derived macrophages (BMDMs) were flushed with complete DMEM. Cell suspensions from peritoneal lavage, spleens and bone marrow were then lysed for removal of red blood cells using ACK lysis buffer (Quality Biological, MD, 118-156-101). Cells were further resuspended in ice cold FACS buffer (2% FBS + 2mM EDTA) in 1x PBS and filtered through 40μm cell strainers to prepare single cell suspension.
Sample Type:Spleen

Treatment:

Treatment ID:TR004390
Treatment Summary:BMDMs were pre-treated with hippuric acid (100 μM) and stimulated with LPS (100 ng/ml) + IFNγ (100 ng/ml) stimuli. BMDMs were treated once for 24 hours. Control samples were untreated BMDMs.

Sample Preparation:

Sampleprep ID:SP004387
Sampleprep Summary:Media was removed from the plates by aspiration and cells were gently washed with chilled sterile PBS. Ice-cold methanol (OmniSolv, EMD Millipore, part # MMX04801) was used to quench and harvest the cells, which were subsequently transferred into glass tubes with PTFE-lined caps (Fisher Scientific 99502-10 and 999815). Samples were spiked with EquiSPLASH LIIDOMIX containing stable isotope-labeled internal standards representing 13 lipid classes (Avanti Polar Lipids, 330731). Lipids were extracted with chloroform/methanol/0.88% sodium chloride 2:1:1, and the aqueous layer was reextracted with synthetic organic phase.

Chromatography:

Chromatography ID:CH005344
Instrument Name:Thermo Vanquish
Column Name:Thermo Accucore C30 (150 x 2.1mm,2.6um)
Column Temperature:50
Flow Gradient:0 to 60% B over 10 min, 60 to 85% B over 10 min, 85 to 100% over 10 min, and holding for 5 min and reequilibrating to initial conditions over 5 min
Flow Rate:0.35 mL/min
Solvent A:50% acetonitrile/50% water; 5 mM ammonium formate; 0.1% formic acid
Solvent B:88% isopropanol/10% acetonitrile/2% water; 5 mM ammonium formate; 0.1% formic acid
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN007039
Analysis Type:MS
Chromatography ID:CH005344
Num Factors:5
Num Metabolites:921
Units:Peak Area
  
Analysis ID:AN007040
Analysis Type:MS
Chromatography ID:CH005344
Num Factors:5
Num Metabolites:273
Units:Peak Area
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