Summary of Study ST002177

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 PR001385. The data can be accessed directly via it's Project DOI: 10.21228/M8PT3H 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.

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Study ID	ST002177
Study Title	Multiomics Analyses Reveal the Central Role of Pentose Phosphate Pathway in Resident Thymic Macrophages to Cope with Efferocytosis-Associated Stress
Study Summary	Tissue-resident macrophages (TRMs) are heterogeneous cell populations found throughout the body. Depending on their location, they perform diverse functions maintaining tissue homeostasis and providing immune surveillance. To survive and function within, TRMs adapt metabolically to the distinct microenvironments. However, little is known about the metabolic signatures of TRMs. The thymus provides a nurturing milieu for developing thymocytes yet efficiently removes those that failed the selection, relying on the TRMs – resident thymic macrophages (TMφs). This study harnesses multiomics analyses to characterize TMφs and unveils their unique metabolic features. We find that the pentose phosphate pathway (PPP) is preferentially activated in TMφs, responding to the reduction-oxidation demands associated with the efferocytosis of dying thymocytes. The blockade of PPP in Mφs leads to decreased efferocytosis, which can be rescued by ROS scavengers. Our study reveals the key role of PPP in TMφs and underscores the importance of metabolic adaptation in supporting Mφ efferocytosis.
Institute	National Yang Ming Chiao Tung University
Department	Institute of Microbiology and Immunology
Laboratory	Chai-Lin Hsu
Last Name	Hsu
First Name	Chia-Lin
Address	R309, Biomedical Building, NYCU, No. 155, Sec. 2, Linong St., Beitou Dist. Taipei 112, Taiwan
Email	clhsu@nycu.edu.tw
Phone	+886-2-2826-7000 ext:65619
Submit Date	2022-05-21
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2022-06-09
Release Version	1

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Project:

Project ID:	PR001385
Project DOI:	doi: 10.21228/M8PT3H
Project Title:	Multiomics Analyses Reveal the Central Role of Pentose Phosphate Pathway in Resident Thymic Macrophages to Cope with Efferocytosis-Associated Stress
Project Summary:	Tissue-resident macrophages (TRMs) are heterogeneous cell populations found throughout the body. Depending on their location, they perform diverse functions maintaining tissue homeostasis and providing immune surveillance. To survive and function within, TRMs adapt metabolically to the distinct microenvironments. However, little is known about the metabolic signatures of TRMs. The thymus provides a nurturing milieu for developing thymocytes yet efficiently removes those that failed the selection, relying on the TRMs – resident thymic macrophages (TMφs). This study harnesses multiomics analyses to characterize TMφs and unveils their unique metabolic features. We find that the pentose phosphate pathway (PPP) is preferentially activated in TMφs, responding to the reduction-oxidation demands associated with the efferocytosis of dying thymocytes. The blockade of PPP in Mφs leads to decreased efferocytosis, which can be rescued by ROS scavengers. Our study reveals the key role of PPP in TMφs and underscores the importance of metabolic adaptation in supporting Mφ efferocytosis.
Institute:	National Yang Ming Chiao Tung University
Department:	Institute of Microbiology and Immunology
Laboratory:	Chia-Lin Hsu
Last Name:	Chia-Lin
First Name:	Hsu
Address:	R309, Biomedical Building, NYCU, No. 155, Sec. 2, Linong St., Beitou Dist. Taipei 112, Taiwan
Email:	clhsu@nycu.edu.tw
Phone:	+886-2-2826-7000 ext: 65619
Publications:	Tsai TL, Zhou TA, Hsieh YT, Wang JC, et al. Multiomics reveal the central role of pentose phosphate pathway in resident thymic macrophages to cope with efferocytosis-associated stress. Cell Rep 2022 Jul 12;40(2):111065. PMID: 35830797 https://doi.org/10.1016/j.celrep.2022.111065
Contributors:	Tsung-Lin Tsai, Ju-Chu Wang, Chen-Hua Huang, Chao-Hsiung Lin, Chia-Lin Hsu

Subject:

Subject ID:	SU002263
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6
Age Or Age Range:	5-8 weeks
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Cell type	Biological repeat
SA209112	PC_N1_45	peritoneal macropahge	1
SA209113	PC_N1_33	peritoneal macropahge	1
SA209114	PC_N1_21	peritoneal macropahge	1
SA209115	PC_N2_46	peritoneal macropahge	2
SA209116	PC_N2_22	peritoneal macropahge	2
SA209117	PC_N2_34	peritoneal macropahge	2
SA209118	PC_N3_47	peritoneal macropahge	3
SA209119	PC_N3_23	peritoneal macropahge	3
SA209120	PC_N3_35	peritoneal macropahge	3
SA209100	Quality control mix_53	QC	-
SA209101	Quality control mix_29	QC	-
SA209102	Quality control mix_41	QC	-
SA209103	THY_N1_49	Thymic macropahge	1
SA209104	THY_N1_37	Thymic macropahge	1
SA209105	THY_N1_25	Thymic macropahge	1
SA209106	THY_N2_51	Thymic macropahge	2
SA209107	THY_N2_27	Thymic macropahge	2
SA209108	THY_N2_39	Thymic macropahge	2
SA209109	THY_N3_40	Thymic macropahge	3
SA209110	THY_N3_52	Thymic macropahge	3
SA209111	THY_N3_28	Thymic macropahge	3

Showing results 1 to 21 of 21

Showing results 1 to 21 of 21

Collection:

Collection ID:	CO002256
Collection Summary:	The thymic macrophages were sorted by F4/80p, CD64p, and CD11bmed. The peritoneal macrophages were sorted by F4/80p, CD11p. The cells were washed with PBS and stored at -80°C. 5*10^5 cells were collected for methanol extraction. Ultra-pure water was used for metabolite reconstitution after removing the methanol.
Sample Type:	Macrophages

Treatment:

Treatment ID:	TR002275
Treatment Summary:	To obtain the resident thymic macrophages, the thymus was harvested from 5 to 8 weeks old C57BL mice, cut into small pieces, and incubated in DMEM containing 0.4 mg/mL collagenase P and 0.4 mg/mL DNase I at 37˚C for 20 min with frequent gentle mixing. The resulting cell suspension was overlaid on the 57% Percoll Plus solution at the volume ratio of 1:1 and centrifuged at 650 x g for 20 min at 4˚C. The cells at the interface were collected and washed with PBS, and re-suspended in 24G2 supernatant at room temperature for 15 min for blocking. The anti-CD64, anti-CD11b, and anti-F4/80 antibody cocktail in FACS buffer (PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA) was added to the sample and allowed incubation on ice for 20 min. At the end of the staining, the cells were centrifuged, washed, and re-suspended in propidium iodide containing FACS buffer. Live singlets with CD64+CD11bloF4/80+ were defined as resident thymic macrophages (TMφs) and sorted by BD FACSMelody with the purity > 95%. The peritoneal cavity macrophages (PCMφs) were collected by intra-peritoneal injected 5 mL of ice-cold complete DMEM, thoroughly rinsed the peritoneal cavity, and re-collected the solution containing the exudate cells. The cells were processed and stained as described above, and the CD11b+F4/80+ cells were identified as PCMφs and harvested.

Treatment ID:

TR002275

Treatment Summary:

To obtain the resident thymic macrophages, the thymus was harvested from 5 to 8 weeks old C57BL mice, cut into small pieces, and incubated in DMEM containing 0.4 mg/mL collagenase P and 0.4 mg/mL DNase I at 37˚C for 20 min with frequent gentle mixing. The resulting cell suspension was overlaid on the 57% Percoll Plus solution at the volume ratio of 1:1 and centrifuged at 650 x g for 20 min at 4˚C. The cells at the interface were collected and washed with PBS, and re-suspended in 24G2 supernatant at room temperature for 15 min for blocking. The anti-CD64, anti-CD11b, and anti-F4/80 antibody cocktail in FACS buffer (PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA) was added to the sample and allowed incubation on ice for 20 min. At the end of the staining, the cells were centrifuged, washed, and re-suspended in propidium iodide containing FACS buffer. Live singlets with CD64+CD11bloF4/80+ were defined as resident thymic macrophages (TMφs) and sorted by BD FACSMelody with the purity > 95%. The peritoneal cavity macrophages (PCMφs) were collected by intra-peritoneal injected 5 mL of ice-cold complete DMEM, thoroughly rinsed the peritoneal cavity, and re-collected the solution containing the exudate cells. The cells were processed and stained as described above, and the CD11b+F4/80+ cells were identified as PCMφs and harvested.

Sample Preparation:

Sampleprep ID:	SP002269
Sampleprep Summary:	Sorted 5 x 105 tissue-resident macrophages were re-suspended in 1 mL LC-MS grade methanol and processed to obtain the metabolites. In brief, the proteins in the sample were precipitated and removed while the supernatant was collected and evaporated by a vacuum concentrator. The resulting metabolite extracts were resolved in 50 µL of ultra-pure water and transferred to a reduced-volume autosampler vial for LC-MS analysis. For liquid chromatography, the ACQUITY BEH C18 column (100 mm length x 2.1 mm internal diameter, 1.7 µm particles) was used and maintained at 40°C in the ultra-performance liquid chromatography (UPLC). Samples were eluted with gradient process at 0.3 mL/min using mobile phase (A) 0.1 % ammonium hydroxide in LC-MS grade water and mobile phase (B) 0.1 % ammonium hydroxide in LC-MS grade acetonitrile (1 % B for 0.5 min, 1–100 % B in 4 min, 100 % B for 0.5 min, 100-1 % B in 1 min, 1 % B for 3 min). The mass spectrometry data were acquired through the Waters Xevo G2-XS QTof in negative mode.

Sampleprep ID:

SP002269

Sampleprep Summary:

Sorted 5 x 105 tissue-resident macrophages were re-suspended in 1 mL LC-MS grade methanol and processed to obtain the metabolites. In brief, the proteins in the sample were precipitated and removed while the supernatant was collected and evaporated by a vacuum concentrator. The resulting metabolite extracts were resolved in 50 µL of ultra-pure water and transferred to a reduced-volume autosampler vial for LC-MS analysis. For liquid chromatography, the ACQUITY BEH C18 column (100 mm length x 2.1 mm internal diameter, 1.7 µm particles) was used and maintained at 40°C in the ultra-performance liquid chromatography (UPLC). Samples were eluted with gradient process at 0.3 mL/min using mobile phase (A) 0.1 % ammonium hydroxide in LC-MS grade water and mobile phase (B) 0.1 % ammonium hydroxide in LC-MS grade acetonitrile (1 % B for 0.5 min, 1–100 % B in 4 min, 100 % B for 0.5 min, 100-1 % B in 1 min, 1 % B for 3 min). The mass spectrometry data were acquired through the Waters Xevo G2-XS QTof in negative mode.

Combined analysis:

Analysis ID	AN003565
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters Acquity BEH C8 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Waters Xevo-G2-XS
Ion Mode	NEGATIVE
Units	peak area

Chromatography:

Chromatography ID:	CH002636
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C8 (100 x 2.1mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003322
Analysis ID:	AN003565
Instrument Name:	Waters Xevo-G2-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Raw data is processed by Progenesis QI software. The features were matched to the KEGG compounds through Chemspider while the mass error was limited to 15 ppm.
Ion Mode:	NEGATIVE