{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002177","ANALYSIS_ID":"AN003565","VERSION":"1","CREATED_ON":"08-10-2022"},

"PROJECT":{"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","DOI":"http://dx.doi.org/10.21228/M8PT3H","CONTRIBUTORS":"Tsung-Lin Tsai, Ju-Chu Wang, Chen-Hua Huang, Chao-Hsiung Lin, Chia-Lin Hsu"},

"STUDY":{"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"},

"SUBJECT":{"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"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"PC_N1_21",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"1"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N1_21.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N1_33",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"1"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N1_33.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N1_45",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"1"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N1_45.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N2_22",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"2"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N2_22.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N2_34",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"2"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N2_34.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N2_46",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"2"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N2_46.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N3_23",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"3"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N3_23.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N3_35",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"3"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N3_35.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"PC_N3_47",
"Factors":{"Cell type":"peritoneal macropahge","Biological repeat":"3"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_pc N3_47.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Quality control mix_29",
"Factors":{"Cell type":"QC","Biological repeat":"-"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_QC_29.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Quality control mix_41",
"Factors":{"Cell type":"QC","Biological repeat":"-"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_QC_41.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"Quality control mix_53",
"Factors":{"Cell type":"QC","Biological repeat":"-"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_QC_53.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N1_25",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"1"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N1_25.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N1_37",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"1"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N1_37.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N1_49",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"1"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N1_49.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N2_27",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"2"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N2_27.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N2_39",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"2"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N2_39.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N2_51",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"2"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N2_51.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N3_28",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"3"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N3_28.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N3_40",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"3"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N3_40.mzXML"}
},
{
"Subject ID":"-",
"Sample ID":"THY_N3_52",
"Factors":{"Cell type":"Thymic macropahge","Biological repeat":"3"},
"Additional sample data":{"RAW_FILE_NAME":"201903015_thy N3_52.mzXML"}
}
],
"COLLECTION":{"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_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."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)","CHROMATOGRAPHY_TYPE":"Reversed phase"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"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","MS_RESULTS_FILE":"ST002177_AN003565_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}