Summary of Study ST004258

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 PR002686. The data can be accessed directly via it's Project DOI: 10.21228/M8FZ77 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 IDST004258
Study TitleMitochondria-localized MLKL channel function drives proinflammatory response independent of necroptosis
Study SummaryExperiment design: BMDMs were isolated from the bone marrow of Mlkl-/-/Mlkl+/+ and Ripk3-/-/Ripk3+/+ mouse femurs and tibias and cultured in DMEM medium (Gibco, C11995500BT, USA) containing 25 ng/mL M-CSF (meilunbio, MA0608, China) and 10% FBS (Gibco, A5669701, USA) for 7 days, with a medium change at 3.5 days. After successful differentiation, cells were incubated with PBS (meilunbio, MA0015, China) or 100 ng/mL LPS (sigma, L2630, USA) for 24 hours. Each experimental group of three biological replicates was performed, with each biological replicate undergoing two technical replicates. As a result, we discovered that deletion of either MLKL or RIPK3 resulted in a comparable reduction of key inflammatory metabolites, including succinate, fumarate, lactate, and citrate. Considering the effects of MLKL and RIPK3 on ROS, MMP, the NF-κB signaling pathway, and metabolomic profiles, we speculate that although MLKL functions in a RIPK3-independent manner, deficiencies in either MLKL or RIPK3 can influence proinflammatory responses and metabolic reprogramming. These processes may be interconnected through multiple feedback regulatory mechanisms.
Institute
Fudan University
DepartmentInstitutes of Biomedical Sciences
LaboratorySystems biology Laboratory
Last NameQie
First NameJingbo
AddressInstitutes of Biomedical Sciences, Fudan University, No. 131, Dong 'an Road, Shanghai 200032, China
Emailjingboqie@fudan.edu.cn
Phone17717890408
Submit Date2025-09-28
Raw Data AvailableYes
Raw Data File Type(s)d
Analysis Type DetailGC-MS
Release Date2025-10-10
Release Version1
Jingbo Qie Jingbo Qie
https://dx.doi.org/10.21228/M8FZ77
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002686
Project DOI:doi: 10.21228/M8FZ77
Project Title:Mitochondria-localized MLKL channel function drives proinflammatory response independent of necroptosis
Project Summary:Mixed lineage kinase domain-like protein (MLKL) has been well-established as an executor of necroptosis. However, its necroptosis independent functions have recently garnered attention, particularly its involvement in non-necroptotic pathological processes. Here, we found that MLKL expression was upregulated in macrophages during inflammation. Our proteomic and functional assays revealed that, despite the absence of necroptosis, metabolic dysregulation and inflammatory responses were still MLKL-dependent. These phenomena were notably suppressed in MLKL-deficient macrophages. Further investigation showed that MLKL translocates to the mitochondrial membrane during inflammation, where its interaction with cardiolipin enhances channel activity, resulting in mitochondrial dysfunction and an amplified inflammatory response. In both in vitro and in vivo models, MLKL deficiency restored mitochondrial function, relieved inflammation, reduced tissue damage. These findings establish MLKL channel as a crucial regulator of macrophage inflammation, independent of its role in necroptosis, and highlight its potential as a therapeutic target in inflammation-associated diseases such as sepsis.
Institute:Fudan University
Department:Institutes of Biomedical Sciences
Laboratory:Systems biology Laboratory
Last Name:Qie
First Name:Jingbo
Address:Institutes of Biomedical Sciences, Fudan University, No. 131, Dong 'an Road, Shanghai 200032, China
Email:jingboqie@fudan.edu.cn
Phone:17717890408

Subject:

Subject ID:SU004411
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:C57BL/6J
Age Or Age Range:8-10 weeks
Gender:Male
Cell Strain Details:Bone-marrow derived macrophages (BMDM)
Cell Primary Immortalized:Primary Cells
Cell Passage Number:1
Cell Counts:1e6

Factors:

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

mb_sample_id local_sample_id experimental factors Sample source
SA496880mlkl-ko pbs2-1ctl1 mlkl-ko
SA496881mlkl-ko pbs3-2ctl1 mlkl-ko
SA496882mlkl-ko pbs3-1ctl1 mlkl-ko
SA496883mlkl-ko pbs2-2ctl1 mlkl-ko
SA496884mlkl-ko pbs1-1ctl1 mlkl-ko
SA496885mlkl-ko pbs1-2ctl1 mlkl-ko
SA496886Ripk3-ko pbs3-2ctl1 ripk3-ko
SA496887Ripk3-ko pbs3-1ctl1 ripk3-ko
SA496888Ripk3-ko pbs2-2ctl1 ripk3-ko
SA496889Ripk3-ko pbs2-1ctl1 ripk3-ko
SA496890Ripk3-ko pbs1-2ctl1 ripk3-ko
SA496891Ripk3-ko pbs1-1ctl1 ripk3-ko
SA496892wt1 pbs1-2ctl1 wt1-mlkl-ko-littermate
SA496893wt1 pbs1-1ctl1 wt1-mlkl-ko-littermate
SA496894wt1 pbs3-1ctl1 wt1-mlkl-ko-littermate
SA496895wt1 pbs2-2ctl1 wt1-mlkl-ko-littermate
SA496896wt1 pbs2-1ctl1 wt1-mlkl-ko-littermate
SA496897wt1 pbs3-2ctl1 wt1-mlkl-ko-littermate
SA496898wt2 pbs1-2ctl1 wt2-ripk3-ko-littermate
SA496899wt2 pbs3-2ctl1 wt2-ripk3-ko-littermate
SA496900wt2 pbs3-1ctl1 wt2-ripk3-ko-littermate
SA496901wt2 pbs2-2ctl1 wt2-ripk3-ko-littermate
SA496902wt2 pbs1-1ctl1 wt2-ripk3-ko-littermate
SA496903wt2 pbs2-1ctl1 wt2-ripk3-ko-littermate
SA496904mlkl-ko lps3-2lps mlkl-ko
SA496905mlkl-ko lps3-1lps mlkl-ko
SA496906mlkl-ko lps2-2lps mlkl-ko
SA496907mlkl-ko lps2-1lps mlkl-ko
SA496908mlkl-ko lps1-2lps mlkl-ko
SA496909mlkl-ko lps1-1lps mlkl-ko
SA496910Ripk3-ko lps3-1lps ripk3-ko
SA496911Ripk3-ko lps2-2lps ripk3-ko
SA496912Ripk3-ko lps2-1lps ripk3-ko
SA496913Ripk3-ko lps1-2lps ripk3-ko
SA496914Ripk3-ko lps1-1lps ripk3-ko
SA496915Ripk3-ko lps3-2lps ripk3-ko
SA496916wt1 lps3-2lps wt1-mlkl-ko-littermate
SA496917wt1 lps1-1lps wt1-mlkl-ko-littermate
SA496918wt1 lps1-2lps wt1-mlkl-ko-littermate
SA496919wt1 lps3-1lps wt1-mlkl-ko-littermate
SA496920wt1 lps2-1lps wt1-mlkl-ko-littermate
SA496921wt1 lps2-2lps wt1-mlkl-ko-littermate
SA496922wt2 lps3-2lps wt2-ripk3-ko-littermate
SA496923wt2 lps3-1lps wt2-ripk3-ko-littermate
SA496924wt2 lps2-2lps wt2-ripk3-ko-littermate
SA496925wt2 lps1-2lps wt2-ripk3-ko-littermate
SA496926wt2 lps1-1lps wt2-ripk3-ko-littermate
SA496927wt2 lps2-1lps wt2-ripk3-ko-littermate
Showing results 1 to 48 of 48

Collection:

Collection ID:CO004404
Collection Summary:BMDMs were isolated from the bone marrow of mouse (Ripk3-ko, mlkl-ko and the littermates) femurs and tibias and cultured in DMEM medium (Gibco, C11995500BT, USA) containing 25 ng/mL M-CSF (meilunbio, MA0608, China) and 10% FBS (Gibco, A5669701, USA) for 7 days, with a medium change at 3.5 days. After successful differentiation, cells were incubated with PBS (meilunbio, MA0015, China) or 100 ng/mL LPS (sigma, L2630, USA) for 24 hours.
Sample Type:Macrophages
Collection Location:bone marrow
Collection Frequency:once per mice
Storage Conditions:-80℃

Treatment:

Treatment ID:TR004420
Treatment Summary:After successful differentiation, cells were incubated with PBS (meilunbio, MA0015, China) or 100 ng/mL LPS (sigma, L2630, USA) for 24 hours.

Sample Preparation:

Sampleprep ID:SP004417
Sampleprep Summary:Post-confluent cells in 6-well plates were homogenized in 0.5 mL chilled 80% (v/v) methanol containing heptadecanoic acid (Sigma, H3500, USA) as an internal standard to monitor batch reproducibility. The samples were centrifuged at 12, 000 rpm for 10 min and the supernatant was transferred to a high recovery glass sampling vial (CNW, VAAP-31509–1232-100) to vacuum dry at room temperature. The residue was oximated with 30 μL pyridine containing 20 mg/ml methoxyamine hydrochloride (Sigma-Aldrich, 226904) at 37°C overnight and further derivatized with 20 μL N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (Sigma-Aldrich, 394882) at 70°C for 30 min.
Processing Storage Conditions:-20℃
Extract Storage:-20℃

Chromatography:

Chromatography ID:CH005383
Chromatography Summary:1 μL aliquot of the derivatized sample was injected into Agilent 7890A gas chromatography coupled with Agilent 5975C mass spectrometer. Separation was achieved on a HP-5ms fused-silica capillary column (30 m × 250 μm i.d.; 5% diphenyl - 95% Methylpolysiloxane bonded and crosslinked) with helium as the carrier gas at a constant flow rate of 1 mL/min through the column.
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30 m x 0.25 mm, 0.25 μm)
Column Temperature:280℃
Flow Gradient:none
Flow Rate:1 mL/min
Solvent A:none
Solvent B:none
Chromatography Type:GC

Analysis:

Analysis ID:AN007086
Analysis Type:MS
Chromatography ID:CH005383
Num Factors:8
Num Metabolites:22
Units:peak area
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