#METABOLOMICS WORKBENCH ttsunglin_20220521_063929 DATATRACK_ID:3264 STUDY_ID:ST002177 ANALYSIS_ID:AN003565 VERSION 1 CREATED_ON 08-10-2022 #PROJECT PR:PROJECT_TITLE Multiomics Analyses Reveal the Central Role of Pentose Phosphate Pathway in PR:PROJECT_TITLE Resident Thymic Macrophages to Cope with Efferocytosis-Associated Stress PR:PROJECT_SUMMARY Tissue-resident macrophages (TRMs) are heterogeneous cell populations found PR:PROJECT_SUMMARY throughout the body. Depending on their location, they perform diverse functions PR:PROJECT_SUMMARY maintaining tissue homeostasis and providing immune surveillance. To survive and PR:PROJECT_SUMMARY function within, TRMs adapt metabolically to the distinct microenvironments. PR:PROJECT_SUMMARY However, little is known about the metabolic signatures of TRMs. The thymus PR:PROJECT_SUMMARY provides a nurturing milieu for developing thymocytes yet efficiently removes PR:PROJECT_SUMMARY those that failed the selection, relying on the TRMs – resident thymic PR:PROJECT_SUMMARY macrophages (TMφs). This study harnesses multiomics analyses to characterize PR:PROJECT_SUMMARY TMφs and unveils their unique metabolic features. We find that the pentose PR:PROJECT_SUMMARY phosphate pathway (PPP) is preferentially activated in TMφs, responding to the PR:PROJECT_SUMMARY reduction-oxidation demands associated with the efferocytosis of dying PR:PROJECT_SUMMARY thymocytes. The blockade of PPP in Mφs leads to decreased efferocytosis, which PR:PROJECT_SUMMARY can be rescued by ROS scavengers. Our study reveals the key role of PPP in TMφs PR:PROJECT_SUMMARY and underscores the importance of metabolic adaptation in supporting Mφ PR:PROJECT_SUMMARY efferocytosis. PR:INSTITUTE National Yang Ming Chiao Tung University PR:DEPARTMENT Institute of Microbiology and Immunology PR:LABORATORY Chia-Lin Hsu PR:LAST_NAME Chia-Lin PR:FIRST_NAME Hsu PR:ADDRESS R309, Biomedical Building, NYCU, No. 155, Sec. 2, Linong St., Beitou Dist. PR:ADDRESS Taipei 112, Taiwan PR:EMAIL clhsu@nycu.edu.tw PR:PHONE +886-2-2826-7000 ext: 65619 PR:PUBLICATIONS Tsai TL, Zhou TA, Hsieh YT, Wang JC, et al. Multiomics reveal the central role PR:PUBLICATIONS of pentose phosphate pathway in resident thymic macrophages to cope with PR:PUBLICATIONS efferocytosis-associated stress. Cell Rep 2022 Jul 12;40(2):111065. PMID: PR:PUBLICATIONS 35830797 https://doi.org/10.1016/j.celrep.2022.111065 PR:DOI http://dx.doi.org/10.21228/M8PT3H PR:CONTRIBUTORS Tsung-Lin Tsai, Ju-Chu Wang, Chen-Hua Huang, Chao-Hsiung Lin, Chia-Lin Hsu #STUDY ST:STUDY_TITLE Multiomics Analyses Reveal the Central Role of Pentose Phosphate Pathway in ST:STUDY_TITLE Resident Thymic Macrophages to Cope with Efferocytosis-Associated Stress ST:STUDY_SUMMARY Tissue-resident macrophages (TRMs) are heterogeneous cell populations found ST:STUDY_SUMMARY throughout the body. Depending on their location, they perform diverse functions ST:STUDY_SUMMARY maintaining tissue homeostasis and providing immune surveillance. To survive and ST:STUDY_SUMMARY function within, TRMs adapt metabolically to the distinct microenvironments. ST:STUDY_SUMMARY However, little is known about the metabolic signatures of TRMs. The thymus ST:STUDY_SUMMARY provides a nurturing milieu for developing thymocytes yet efficiently removes ST:STUDY_SUMMARY those that failed the selection, relying on the TRMs – resident thymic ST:STUDY_SUMMARY macrophages (TMφs). This study harnesses multiomics analyses to characterize ST:STUDY_SUMMARY TMφs and unveils their unique metabolic features. We find that the pentose ST:STUDY_SUMMARY phosphate pathway (PPP) is preferentially activated in TMφs, responding to the ST:STUDY_SUMMARY reduction-oxidation demands associated with the efferocytosis of dying ST:STUDY_SUMMARY thymocytes. The blockade of PPP in Mφs leads to decreased efferocytosis, which ST:STUDY_SUMMARY can be rescued by ROS scavengers. Our study reveals the key role of PPP in TMφs ST:STUDY_SUMMARY and underscores the importance of metabolic adaptation in supporting Mφ ST:STUDY_SUMMARY efferocytosis. ST:INSTITUTE National Yang Ming Chiao Tung University ST:DEPARTMENT Institute of Microbiology and Immunology ST:LABORATORY Chai-Lin Hsu ST:LAST_NAME Hsu ST:FIRST_NAME Chia-Lin ST:ADDRESS R309, Biomedical Building, NYCU, No. 155, Sec. 2, Linong St., Beitou Dist. ST:ADDRESS Taipei 112, Taiwan ST:EMAIL clhsu@nycu.edu.tw ST:PHONE +886-2-2826-7000 ext:65619 ST:SUBMIT_DATE 2022-05-21 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:AGE_OR_AGE_RANGE 5-8 weeks SU:GENDER Male and female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - PC_N1_21 Cell type:peritoneal macropahge | Biological repeat:1 RAW_FILE_NAME=201903015_pc N1_21.mzXML SUBJECT_SAMPLE_FACTORS - PC_N1_33 Cell type:peritoneal macropahge | Biological repeat:1 RAW_FILE_NAME=201903015_pc N1_33.mzXML SUBJECT_SAMPLE_FACTORS - PC_N1_45 Cell type:peritoneal macropahge | Biological repeat:1 RAW_FILE_NAME=201903015_pc N1_45.mzXML SUBJECT_SAMPLE_FACTORS - PC_N2_22 Cell type:peritoneal macropahge | Biological repeat:2 RAW_FILE_NAME=201903015_pc N2_22.mzXML SUBJECT_SAMPLE_FACTORS - PC_N2_34 Cell type:peritoneal macropahge | Biological repeat:2 RAW_FILE_NAME=201903015_pc N2_34.mzXML SUBJECT_SAMPLE_FACTORS - PC_N2_46 Cell type:peritoneal macropahge | Biological repeat:2 RAW_FILE_NAME=201903015_pc N2_46.mzXML SUBJECT_SAMPLE_FACTORS - PC_N3_23 Cell type:peritoneal macropahge | Biological repeat:3 RAW_FILE_NAME=201903015_pc N3_23.mzXML SUBJECT_SAMPLE_FACTORS - PC_N3_35 Cell type:peritoneal macropahge | Biological repeat:3 RAW_FILE_NAME=201903015_pc N3_35.mzXML SUBJECT_SAMPLE_FACTORS - PC_N3_47 Cell type:peritoneal macropahge | Biological repeat:3 RAW_FILE_NAME=201903015_pc N3_47.mzXML SUBJECT_SAMPLE_FACTORS - Quality control mix_29 Cell type:QC | Biological repeat:- RAW_FILE_NAME=201903015_QC_29.mzXML SUBJECT_SAMPLE_FACTORS - Quality control mix_41 Cell type:QC | Biological repeat:- RAW_FILE_NAME=201903015_QC_41.mzXML SUBJECT_SAMPLE_FACTORS - Quality control mix_53 Cell type:QC | Biological repeat:- RAW_FILE_NAME=201903015_QC_53.mzXML SUBJECT_SAMPLE_FACTORS - THY_N1_25 Cell type:Thymic macropahge | Biological repeat:1 RAW_FILE_NAME=201903015_thy N1_25.mzXML SUBJECT_SAMPLE_FACTORS - THY_N1_37 Cell type:Thymic macropahge | Biological repeat:1 RAW_FILE_NAME=201903015_thy N1_37.mzXML SUBJECT_SAMPLE_FACTORS - THY_N1_49 Cell type:Thymic macropahge | Biological repeat:1 RAW_FILE_NAME=201903015_thy N1_49.mzXML SUBJECT_SAMPLE_FACTORS - THY_N2_27 Cell type:Thymic macropahge | Biological repeat:2 RAW_FILE_NAME=201903015_thy N2_27.mzXML SUBJECT_SAMPLE_FACTORS - THY_N2_39 Cell type:Thymic macropahge | Biological repeat:2 RAW_FILE_NAME=201903015_thy N2_39.mzXML SUBJECT_SAMPLE_FACTORS - THY_N2_51 Cell type:Thymic macropahge | Biological repeat:2 RAW_FILE_NAME=201903015_thy N2_51.mzXML SUBJECT_SAMPLE_FACTORS - THY_N3_28 Cell type:Thymic macropahge | Biological repeat:3 RAW_FILE_NAME=201903015_thy N3_28.mzXML SUBJECT_SAMPLE_FACTORS - THY_N3_40 Cell type:Thymic macropahge | Biological repeat:3 RAW_FILE_NAME=201903015_thy N3_40.mzXML SUBJECT_SAMPLE_FACTORS - THY_N3_52 Cell type:Thymic macropahge | Biological repeat:3 RAW_FILE_NAME=201903015_thy N3_52.mzXML #COLLECTION CO:COLLECTION_SUMMARY The thymic macrophages were sorted by F4/80p, CD64p, and CD11bmed. The CO:COLLECTION_SUMMARY peritoneal macrophages were sorted by F4/80p, CD11p. The cells were washed with CO:COLLECTION_SUMMARY PBS and stored at -80°C. 5*10^5 cells were collected for methanol extraction. CO:COLLECTION_SUMMARY Ultra-pure water was used for metabolite reconstitution after removing the CO:COLLECTION_SUMMARY methanol. CO:SAMPLE_TYPE Macrophages #TREATMENT TR:TREATMENT_SUMMARY To obtain the resident thymic macrophages, the thymus was harvested from 5 to 8 TR:TREATMENT_SUMMARY weeks old C57BL mice, cut into small pieces, and incubated in DMEM containing TR:TREATMENT_SUMMARY 0.4 mg/mL collagenase P and 0.4 mg/mL DNase I at 37˚C for 20 min with frequent TR:TREATMENT_SUMMARY gentle mixing. The resulting cell suspension was overlaid on the 57% Percoll TR:TREATMENT_SUMMARY Plus solution at the volume ratio of 1:1 and centrifuged at 650 x g for 20 min TR:TREATMENT_SUMMARY at 4˚C. The cells at the interface were collected and washed with PBS, and TR:TREATMENT_SUMMARY re-suspended in 24G2 supernatant at room temperature for 15 min for blocking. TR:TREATMENT_SUMMARY The anti-CD64, anti-CD11b, and anti-F4/80 antibody cocktail in FACS buffer (PBS TR:TREATMENT_SUMMARY with 0.5% bovine serum albumin (BSA) and 2 mM EDTA) was added to the sample and TR:TREATMENT_SUMMARY allowed incubation on ice for 20 min. At the end of the staining, the cells were TR:TREATMENT_SUMMARY centrifuged, washed, and re-suspended in propidium iodide containing FACS TR:TREATMENT_SUMMARY buffer. Live singlets with CD64+CD11bloF4/80+ were defined as resident thymic TR:TREATMENT_SUMMARY macrophages (TMφs) and sorted by BD FACSMelody with the purity > 95%. The TR:TREATMENT_SUMMARY peritoneal cavity macrophages (PCMφs) were collected by intra-peritoneal TR:TREATMENT_SUMMARY injected 5 mL of ice-cold complete DMEM, thoroughly rinsed the peritoneal TR:TREATMENT_SUMMARY cavity, and re-collected the solution containing the exudate cells. The cells TR:TREATMENT_SUMMARY were processed and stained as described above, and the CD11b+F4/80+ cells were TR:TREATMENT_SUMMARY identified as PCMφs and harvested. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sorted 5 x 105 tissue-resident macrophages were re-suspended in 1 mL LC-MS grade SP:SAMPLEPREP_SUMMARY methanol and processed to obtain the metabolites. In brief, the proteins in the SP:SAMPLEPREP_SUMMARY sample were precipitated and removed while the supernatant was collected and SP:SAMPLEPREP_SUMMARY evaporated by a vacuum concentrator. The resulting metabolite extracts were SP:SAMPLEPREP_SUMMARY resolved in 50 µL of ultra-pure water and transferred to a reduced-volume SP:SAMPLEPREP_SUMMARY autosampler vial for LC-MS analysis. For liquid chromatography, the ACQUITY BEH SP:SAMPLEPREP_SUMMARY C18 column (100 mm length x 2.1 mm internal diameter, 1.7 µm particles) was SP:SAMPLEPREP_SUMMARY used and maintained at 40°C in the ultra-performance liquid chromatography SP:SAMPLEPREP_SUMMARY (UPLC). Samples were eluted with gradient process at 0.3 mL/min using mobile SP:SAMPLEPREP_SUMMARY phase (A) 0.1 % ammonium hydroxide in LC-MS grade water and mobile phase (B) 0.1 SP:SAMPLEPREP_SUMMARY % ammonium hydroxide in LC-MS grade acetonitrile (1 % B for 0.5 min, 1–100 % B SP:SAMPLEPREP_SUMMARY in 4 min, 100 % B for 0.5 min, 100-1 % B in 1 min, 1 % B for 3 min). The mass SP:SAMPLEPREP_SUMMARY spectrometry data were acquired through the Waters Xevo G2-XS QTof in negative SP:SAMPLEPREP_SUMMARY mode. #CHROMATOGRAPHY CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C8 (100 x 2.1mm, 1.7um) CH:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Xevo-G2-XS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:MS_COMMENTS Raw data is processed by Progenesis QI software. The features were matched to MS:MS_COMMENTS the KEGG compounds through Chemspider while the mass error was limited to 15 MS:MS_COMMENTS ppm. MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST002177_AN003565_Results.txt UNITS:peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END