Summary of Study ST003588

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 PR002218. The data can be accessed directly via it's Project DOI: 10.21228/M8XF95 This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.

Study ID	ST003588
Study Title	Mitochondria complex III is essential for IL-10 secretion in macrophages independent of respiration
Study Summary	Mitochondrial electron transport chain (ETC) function is linked to macrophage function, however, mechanisms underlying mitochondrial ETC control of macrophage immune responses are not fully understood. We used genetic tools to examine the necessity of mitochondrial electron transport chain (ETC)-dependent respiration and the production of reactive oxygen species (mtROS) in macrophage immune responses. Here we report that mitochondrial ETC complex III (CIII)-deficient mouse macrophages, which have impaired macrophage respiration and mtROS production, exhibit increased susceptibility to influenza A virus and LPS-induced endotoxic shock. Mitochondrial CIII-deficient bone marrow-derived macrophages (BMDMs) produced IL-10 but exhibit dampened release following TLR3 or TLR4 stimulation in vitro. Surprisingly, restoring mitochondrial respiration without generating mtROS in mitochondrial CIII-deficient macrophages with Ciona intestinalis alternative oxidase (AOX) is not sufficient to reverse the increased vulnerability to LPS-induced endotoxic shock or rescue IL-10 release in vitro. However, activation of PKA, a mtROS-responsive pathway, was sufficient to rescue BMDM IL-10 release following lipopolysaccharide (LPS) stimulation. Additionally, mitochondrial CIII impairment in macrophages does not affect canonical responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight the necessity of mitochondrial ROS but not respiration in the release of IL-10.
Institute	Northwestern University, Feinberg School of Medicine
Last Name	Stoolman
First Name	Joshua
Address	303 E Superior Street
Email	joshua.stoolman@northwestern.edu
Phone	7343559440
Submit Date	2024-10-29
Num Groups	15
Total Subjects	11
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-12-20
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002218
Project DOI:	doi: 10.21228/M8XF95
Project Title:	Mitochondria complex III is essential for IL-10 secretion in macrophages independent of respiration
Project Summary:	Mitochondrial electron transport chain (ETC) function is linked to macrophage function. However, mechanisms underlying mitochondrial ETC control of macrophage immune responses are not fully understood. We used genetic tools to examine the necessity of mitochondrial electron transport chain (ETC)-dependent respiration and the production of reactive oxygen species (mtROS) in macrophage immune responses. Here we report that mitochondrial ETC complex III (CIII)-deficient mouse macrophages, which have impaired macrophage respiration and mtROS production, exhibit increased susceptibility to influenza A virus and LPS-induced endotoxic shock. Mitochondrial CIII-deficient bone marrow-derived macrophages (BMDMs) produced IL-10 but exhibit dampened release following TLR3 or TLR4 stimulation in vitro. Surprisingly, restoring mitochondrial respiration without generating mtROS in mitochondrial CIII-deficient macrophages with Ciona intestinalis alternative oxidase (AOX) is not sufficient to reverse the increased vulnerability to LPS-induced endotoxic shock or rescue IL-10 release in vitro. However, activation of PKA, an mtROS-responsive pathway2, was sufficient to rescue BMDM IL-10 release following LPS stimulation. Additionally, mitochondrial CIII impairment in macrophages does not affect canonical responses to interleukin-4 (IL-4) stimulation. Thus, our results highlight necessity of mitochondrial ROS but not respiration in release of IL-10.
Institute:	Northwestern University, Feinberg School of Medicine
Department:	Pulmonary
Laboratory:	Chandel
Last Name:	Stoolman
First Name:	Joshua
Address:	303 E Superior Street
Email:	joshua.stoolman@northwestern.edu
Phone:	7343559440

Subject:

Subject ID:	SU003717
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Species Group:	Mammals

Factors:

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

mb_sample_id	local_sample_id	Sample source	Genotype	Time	Treatment
SA391553	Cha-Jos-20231019-02	Bone marrow derived macrophages	CTRL	0h	Vehicle
SA391554	Cha-Jos-20231019-35	Bone marrow derived macrophages	CTRL	0h	Vehicle
SA391555	Cha-Jos-20231019-32	Bone marrow derived macrophages	CTRL	0h	Vehicle
SA391556	Cha-Jos-20231019-31	Bone marrow derived macrophages	CTRL	0h	Vehicle
SA391557	Cha-Jos-20231019-01	Bone marrow derived macrophages	CTRL	0h	Vehicle
SA391558	Cha-Jos-20231019-42	Bone marrow derived macrophages	CTRL	1h	LPS
SA391559	Cha-Jos-20231019-45	Bone marrow derived macrophages	CTRL	1h	LPS
SA391560	Cha-Jos-20231019-41	Bone marrow derived macrophages	CTRL	1h	LPS
SA391561	Cha-Jos-20231019-13	Bone marrow derived macrophages	CTRL	1h	LPS
SA391562	Cha-Jos-20231019-14	Bone marrow derived macrophages	CTRL	1h	LPS
SA391563	Cha-Jos-20231019-36	Bone marrow derived macrophages	CTRL	1h	Vehicle
SA391564	Cha-Jos-20231019-37	Bone marrow derived macrophages	CTRL	1h	Vehicle
SA391565	Cha-Jos-20231019-40	Bone marrow derived macrophages	CTRL	1h	Vehicle
SA391566	Cha-Jos-20231019-07	Bone marrow derived macrophages	CTRL	1h	Vehicle
SA391567	Cha-Jos-20231019-08	Bone marrow derived macrophages	CTRL	1h	Vehicle
SA391568	Cha-Jos-20231019-51	Bone marrow derived macrophages	CTRL	2h	LPS
SA391569	Cha-Jos-20231019-52	Bone marrow derived macrophages	CTRL	2h	LPS
SA391570	Cha-Jos-20231019-55	Bone marrow derived macrophages	CTRL	2h	LPS
SA391571	Cha-Jos-20231019-25	Bone marrow derived macrophages	CTRL	2h	LPS
SA391572	Cha-Jos-20231019-26	Bone marrow derived macrophages	CTRL	2h	LPS
SA391573	Cha-Jos-20231019-46	Bone marrow derived macrophages	CTRL	2h	Vehicle
SA391574	Cha-Jos-20231019-47	Bone marrow derived macrophages	CTRL	2h	Vehicle
SA391575	Cha-Jos-20231019-19	Bone marrow derived macrophages	CTRL	2h	Vehicle
SA391576	Cha-Jos-20231019-20	Bone marrow derived macrophages	CTRL	2h	Vehicle
SA391577	Cha-Jos-20231019-50	Bone marrow derived macrophages	CTRL	2h	Vehicle
SA391578	Cha-Jos-20231019-05	Bone marrow derived macrophages	KO + AOX	0h	Vehicle
SA391579	Cha-Jos-20231019-34	Bone marrow derived macrophages	KO + AOX	0h	Vehicle
SA391580	Cha-Jos-20231019-04	Bone marrow derived macrophages	KO + AOX	0h	Vehicle
SA391581	Cha-Jos-20231019-44	Bone marrow derived macrophages	KO + AOX	1h	LPS
SA391582	Cha-Jos-20231019-16	Bone marrow derived macrophages	KO + AOX	1h	LPS
SA391583	Cha-Jos-20231019-17	Bone marrow derived macrophages	KO + AOX	1h	LPS
SA391584	Cha-Jos-20231019-10	Bone marrow derived macrophages	KO + AOX	1h	Vehicle
SA391585	Cha-Jos-20231019-11	Bone marrow derived macrophages	KO + AOX	1h	Vehicle
SA391586	Cha-Jos-20231019-39	Bone marrow derived macrophages	KO + AOX	1h	Vehicle
SA391587	Cha-Jos-20231019-29	Bone marrow derived macrophages	KO + AOX	2h	LPS
SA391588	Cha-Jos-20231019-28	Bone marrow derived macrophages	KO + AOX	2h	LPS
SA391589	Cha-Jos-20231019-54	Bone marrow derived macrophages	KO + AOX	2h	LPS
SA391590	Cha-Jos-20231019-23	Bone marrow derived macrophages	KO + AOX	2h	Vehicle
SA391591	Cha-Jos-20231019-22	Bone marrow derived macrophages	KO + AOX	2h	Vehicle
SA391592	Cha-Jos-20231019-49	Bone marrow derived macrophages	KO + AOX	2h	Vehicle
SA391593	Cha-Jos-20231019-33	Bone marrow derived macrophages	KO	0h	Vehicle
SA391594	Cha-Jos-20231019-03	Bone marrow derived macrophages	KO	0h	Vehicle
SA391595	Cha-Jos-20231019-06	Bone marrow derived macrophages	KO	0h	Vehicle
SA391596	Cha-Jos-20231019-15	Bone marrow derived macrophages	KO	1h	LPS
SA391597	Cha-Jos-20231019-43	Bone marrow derived macrophages	KO	1h	LPS
SA391598	Cha-Jos-20231019-18	Bone marrow derived macrophages	KO	1h	LPS
SA391599	Cha-Jos-20231019-12	Bone marrow derived macrophages	KO	1h	Vehicle
SA391600	Cha-Jos-20231019-38	Bone marrow derived macrophages	KO	1h	Vehicle
SA391601	Cha-Jos-20231019-09	Bone marrow derived macrophages	KO	1h	Vehicle
SA391602	Cha-Jos-20231019-53	Bone marrow derived macrophages	KO	2h	LPS
SA391603	Cha-Jos-20231019-27	Bone marrow derived macrophages	KO	2h	LPS
SA391604	Cha-Jos-20231019-30	Bone marrow derived macrophages	KO	2h	LPS
SA391605	Cha-Jos-20231019-48	Bone marrow derived macrophages	KO	2h	Vehicle
SA391606	Cha-Jos-20231019-21	Bone marrow derived macrophages	KO	2h	Vehicle
SA391607	Cha-Jos-20231019-24	Bone marrow derived macrophages	KO	2h	Vehicle

Showing results 1 to 55 of 55

Showing results 1 to 55 of 55

Collection:

Collection ID:	CO003710
Collection Summary:	After treatment, cells were washed two times with ice-cold saline and scraped into 1mL ice-cold methanol (80% methanol/20% H2O) and stored at -80C overnight. Samples were lysed by 3x cycles of freeze thaw in liquid N2 followed by 37C water bath. Samples were then spun at 16,000xg for 15min. Supernatant was collected and analyzed as below.
Sample Type:	Bone marrow derived Macrophages (BMDMs)

Treatment:

Treatment ID:	TR003726
Treatment Summary:	Glucose-free RPMI (Fisher) supplemented with 10% FBS and 11mM D-Glucose (U-¹³C₆) (Cambridge Isotope Laboratories). BMDMs were plated 2e6 cells per well in 12-well plates and washed with blank Glucose-free RPMI twice prior to treatment plating in Glucose-free RPMI (Fisher) supplemented with 10% FBS and 11mM D-Glucose (U-¹³C₆) (Cambridge Isotope Laboratories). heavy-labeled and treatment with either Lipopolysaccharide (LPS) (100ng/mL, Sigma) or vehicle control for 0, 1 or 2 hours. After treatment, cells were washed two times with ice-cold saline and scraped into 1mL ice-cold methanol (80% methanol/20% H2O) and stored at -80C overnight. Samples were lysed by 3x cycles of freeze thaw in liquid N2 followed by 37C water bath. Samples were then spun at 16,000xg for 15min. Supernatant was collected and analyzed as below.

Treatment ID:

TR003726

Treatment Summary:

Glucose-free RPMI (Fisher) supplemented with 10% FBS and 11mM D-Glucose (U-¹³C₆) (Cambridge Isotope Laboratories). BMDMs were plated 2e6 cells per well in 12-well plates and washed with blank Glucose-free RPMI twice prior to treatment plating in Glucose-free RPMI (Fisher) supplemented with 10% FBS and 11mM D-Glucose (U-¹³C₆) (Cambridge Isotope Laboratories). heavy-labeled and treatment with either Lipopolysaccharide (LPS) (100ng/mL, Sigma) or vehicle control for 0, 1 or 2 hours. After treatment, cells were washed two times with ice-cold saline and scraped into 1mL ice-cold methanol (80% methanol/20% H2O) and stored at -80C overnight. Samples were lysed by 3x cycles of freeze thaw in liquid N2 followed by 37C water bath. Samples were then spun at 16,000xg for 15min. Supernatant was collected and analyzed as below.

Sample Preparation:

Sampleprep ID:	SP003724
Sampleprep Summary:	BMDMs were plated 2e6 cells per well in 12-well plates and washed with blank Glucose-free RPMI twice prior to plating in Glucose-free RPMI (Fisher) supplemented with 10% FBS and 11mM D-Glucose (U-¹³C₆) and subsequent treatment with Lipopolysaccharide LPS (100ng/mL, Sigma) or vehicle control for times indicated in text. After treatment, cells were washed two times with ice-cold saline and scraped into 1mL ice-cold methanol (80% methanol/20% H2O) and stored at -80C overnight. Samples were lysed by 3x cycles of freeze-thaw in liquid N2 followed by a 37C water bath. Samples were then spun at 16,000xg for 15min and supernatants were collected for analysis.

Sampleprep ID:

SP003724

Sampleprep Summary:

BMDMs were plated 2e6 cells per well in 12-well plates and washed with blank Glucose-free RPMI twice prior to plating in Glucose-free RPMI (Fisher) supplemented with 10% FBS and 11mM D-Glucose (U-¹³C₆) and subsequent treatment with Lipopolysaccharide LPS (100ng/mL, Sigma) or vehicle control for times indicated in text. After treatment, cells were washed two times with ice-cold saline and scraped into 1mL ice-cold methanol (80% methanol/20% H2O) and stored at -80C overnight. Samples were lysed by 3x cycles of freeze-thaw in liquid N2 followed by a 37C water bath. Samples were then spun at 16,000xg for 15min and supernatants were collected for analysis.

Combined analysis:

Analysis ID	AN005892
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Dionex Ultimate 3000
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap
Ion Mode	UNSPECIFIED
Units	Peak Area

Chromatography:

Chromatography ID:	CH004475
Chromatography Summary:	15μl aliquot of the sample was used for high-resolution HPLC-tandem mass spectrometry. High-resolution HPLC-tandem mass spectrometry was performed on a Q-Exactive (ThermoFisher Scientific) in line with an electrospray source and an UltiMate 3000 (ThermoFisher Scientific) series HPLC consisting of a binary pump, degasser and autosampler outfitted with a XBridge Amide column (Waters; 4.6 mm × 100 mm dimension and a 3.5 μm particle size). Mobile phase A contained water and acetonitrile (95/5, v/v), 10 mM ammonium hydroxide and 10 mM ammonium acetate (pH 9.0). Mobile phase B was 100% acetonitrile. The gradient was set to 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1–18 min, 75% A; 18–25 min, 15% A, with a flow rate of 400 μl min–1. The capillary of the electrospray ionization source was set to 275 °C, with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. A mass/charge ratio scan ranging from 70 to 850 was used in positive/negative polarity switching mode. MS1 data were collected at a resolution The automatic gain control (AGC) target was set at 1 × 106, with a maximum injection time of 200 ms. The top five precursor ions were fragmented using the higher-energy collisional dissociation cell with normalized collision energy of 30% in MS2 at a resolution of 17,500. Data were acquired with Xcalibur software (v.4.1; ThermoFisher Scientific).
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Column Temperature:	25
Flow Gradient:	0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1–18 min, 75% A; 18–25 min, 15% A
Flow Rate:	400 uL/min
Injection Temperature:	275
Solvent A:	95% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate (pH 9.0)
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS005610
Analysis ID:	AN005892
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	A 15μl aliquot of the sample was used for high-resolution HPLC-tandem mass spectrometry. High-resolution HPLC-tandem mass spectrometry was performed on a Q-Exactive (ThermoFisher Scientific) in line with an electrospray source and an UltiMate 3000 (ThermoFisher Scientific) series HPLC consisting of a binary pump, degasser and autosampler outfitted with a Xbridge Amide column (Waters; 3 mm × 100 mm dimension and a 3.5 μm particle size). Mobile phase A contained water and acetonitrile (95/5, v/v), 10 mM ammonium hydroxide and 10 mM ammonium acetate (pH 9.0). Mobile phase B was 100% acetonitrile. The gradient was set to 0 min, 15% A; 2.5 min, 30% A; 7 min, 43% A; 16 min, 62% A; 16.1–18 min, 75% A; 18–25 min, 15% A, with a flow rate of 400 μl min–1. The capillary of the electrospray ionization source was set to 275 °C, with sheath gas at 45 arbitrary units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. A mass/charge ratio scan ranging from 70 to 850 was used in positive/negative polarity switching mode. MS1 data were collected at a resolution The automatic gain control (AGC) target was set at 1 × 106, with a maximum injection time of 200 ms. The top five precursor ions were fragmented using the higher-energy collisional dissociation cell with normalized collision energy of 30% in MS2 at a resolution of 17,500. Data were acquired with Xcalibur software (v.4.1; ThermoFisher Scientific).
Ion Mode:	UNSPECIFIED