Summary of Study ST002974
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 PR001851. The data can be accessed directly via it's Project DOI: 10.21228/M8FT6G 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 | ST002974 |
| Study Title | Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In Cutaneous Lesions |
| Study Type | Untargeted MS |
| Study Summary | Cutaneous leishmaniasis is characterized by extensive skin lesions, which are usually painless despite being associated with extensive inflammation. The molecular mechanisms responsible for this analgesia have not been identified. Through untargeted metabolomics, we found enriched anti-nociceptive metabolic pathways in L. mexicana-infected mice. Purines were elevated in infected macrophages and at the lesion site during chronic infection. These purines have anti-inflammatory and analgesic properties by acting through adenosine receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also found arachidonic acid metabolism enriched in the ear lesions compared to the non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites. |
| Institute | The Ohio State University |
| Department | Pathology and Microbiology |
| Last Name | Satoskar |
| First Name | Abhay |
| Address | Evans Hall, 520 King Avenue, Columbus, OH, 43201, USA |
| abhay.satoskar@osumc.edu | |
| Phone | (614) 293-0537 |
| Submit Date | 2023-10-17 |
| Analysis Type Detail | LC-MS |
| Release Date | 2024-10-18 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001851 |
| Project DOI: | doi: 10.21228/M8FT6G |
| Project Title: | Leishmania mexicana Promotes Pain-reducing Metabolomic Reprogramming In Cutaneous Lesions |
| Project Type: | Untargeted MS |
| Project Summary: | Cutaneous leishmaniasis is characterized by extensive skin lesions, which are usually painless despite being associated with extensive inflammation. The molecular mechanisms responsible for this analgesia have not been identified. Through untargeted metabolomics, we found enriched anti-nociceptive metabolic pathways in L. mexicana-infected mice. Purines were elevated in infected macrophages and at the lesion site during chronic infection. These purines have anti-inflammatory and analgesic properties by acting through adenosine receptors, inhibiting TRPV1 channels, and promoting IL-10 production. We also found arachidonic acid metabolism enriched in the ear lesions compared to the non-infected controls. Arachidonic acid is a metabolite of anandamide (AEA) and 2-arachidonoylglycerol (2-AG). These endocannabinoids act on cannabinoid receptors 1 and 2 and TRPV1 channels to exert anti-inflammatory and analgesic effects. Our study provides evidence of metabolic pathways upregulated during L. mexicana infection that may mediate anti-nociceptive effects experienced by CL patients and identifies macrophages as a source of these metabolites. |
| Institute: | The Ohio State University |
| Department: | Pathology and Microbiology |
| Last Name: | Satoskar |
| First Name: | Abhay |
| Address: | Evans Hall, 520 King Avenue, Columbus, OH, 43201, USA |
| Email: | abhay.satoskar@osumc.edu |
| Phone: | (614) 293-0537 |
Subject:
| Subject ID: | SU003087 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Gender: | Female |
Factors:
Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample Type |
|---|---|---|
| SA323106 | RAW-N_Cell_1 | Cell pellet derived from naïve RAW 264.7. Technical replicate 1 |
| SA323107 | RAW-N_Cell_2 | Cell pellet derived from naïve RAW 264.7. Technical replicate 2 |
| SA323108 | RAW-N_Cell_3 | Cell pellet derived from naïve RAW 264.7. Technical replicate 3 |
| SA323097 | RAW-L1_Cell_1 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 1 |
| SA323098 | RAW-L1_Cell_2 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 2 |
| SA323099 | RAW-L1_Cell_3 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 1, technical replicate 3 |
| SA323100 | RAW-L2_Cell_1 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 1 |
| SA323101 | RAW-L2_Cell_2 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 2 |
| SA323102 | RAW-L2_Cell_3 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 2, technical replicate 3 |
| SA323103 | RAW-L3_Cell_1 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 1 |
| SA323104 | RAW-L3_Cell_2 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 2 |
| SA323105 | RAW-L3_Cell_3 | Cell pellet derived from RAW 264.7 infected with L. mexicana. Biological replicate 3, technical replicate 3 |
| SA323109 | BMDMPellet-LMex1_1 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 1 |
| SA323110 | BMDMPellet-LMex1_2 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 2 |
| SA323111 | BMDMPellet-LMex1_3 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 3 |
| SA323112 | BMDMPellet-LMex2_1 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 1 |
| SA323113 | BMDMPellet-LMex2_2 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 2 |
| SA323114 | BMDMPellet-LMex2_3 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 3 |
| SA323115 | BMDMPellet-LMex3_1 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 1 |
| SA323116 | BMDMPellet-LMex3_2 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 2 |
| SA323117 | BMDMPellet-LMex3_3 | Cell pellet from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 3 |
| SA323118 | BMDMPellet-Control1_1 | Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 1 |
| SA323119 | BMDMPellet-Control1_2 | Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 2 |
| SA323120 | BMDMPellet-Control1_3 | Cell pellet from naïve BMDMs. Biological replicate 1, technical replicate 3 |
| SA323121 | BMDMPellet-Control2_1 | Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 1 |
| SA323122 | BMDMPellet-Control2_2 | Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 2 |
| SA323123 | BMDMPellet-Control2_3 | Cell pellet from naïve BMDMs. Biological replicate 2, technical replicate 3 |
| SA323124 | BMDMPellet-Control3_1 | Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 1 |
| SA323125 | BMDMPellet-Control3_2 | Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 2 |
| SA323126 | BMDMPellet-Control3_3 | Cell pellet from naïve BMDMs. Biological replicate 3, technical replicate 3 |
| SA323127 | BMDMSup-LMex1_1 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 1 |
| SA323128 | BMDMSup-LMex1_2 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 2 |
| SA323129 | BMDMSup-LMex1_3 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 1, technical replicate 3 |
| SA323130 | BMDMSup-LMex2_1 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 1 |
| SA323131 | BMDMSup-LMex2_2 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 2 |
| SA323132 | BMDMSup-LMex2_3 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 2, technical replicate 3 |
| SA323133 | BMDMSup-LMex3_1 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 1 |
| SA323134 | BMDMSup-LMex3_2 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 2 |
| SA323135 | BMDMSup-LMex3_3 | Supernatant from BMDMs infected with L. mexicana. Biological replicate 3, technical replicate 3 |
| SA323136 | BMDMSup-Control1_1 | Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 1 |
| SA323137 | BMDMSup-Control1_2 | Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 2 |
| SA323138 | BMDMSup-Control1_3 | Supernatant from naïve BMDMs. Biological replicate 1, technical replicate 3 |
| SA323139 | BMDMSup-Control2_1 | Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 1 |
| SA323140 | BMDMSup-Control2_2 | Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 2 |
| SA323141 | BMDMSup-Control2_3 | Supernatant from naïve BMDMs. Biological replicate 2, technical replicate 3 |
| SA323142 | BMDMSup-Control3_1 | Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 1 |
| SA323143 | BMDMSup-Control3_2 | Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 2 |
| SA323144 | BMDMSup-Control3_3 | Supernatant from naïve BMDMs. Biological replicate 3, technical replicate 3 |
| SA323145 | VolpedoTissue_N1_1 | Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 1 |
| SA323146 | VolpedoTissue_N1_2 | Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 2 |
| SA323147 | VolpedoTissue_N1_3 | Tissue from the ear of a naïve mouse. Biological replicate 1, technical replicate 3 |
| SA323148 | VolpedoTissue_N2_1 | Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 1 |
| SA323149 | VolpedoTissue_N2_2 | Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 2 |
| SA323150 | VolpedoTissue_N2_3 | Tissue from the ear of a naïve mouse. Biological replicate 2, technical replicate 3 |
| SA323151 | VolpedoTissue_Ipsi1_1 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 1 |
| SA323152 | VolpedoTissue_Ipsi1_2 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 2 |
| SA323153 | VolpedoTissue_Ipsi1_3 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 1, technical replicate 3 |
| SA323154 | VolpedoTissue_Ipsi2_1 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 1 |
| SA323155 | VolpedoTissue_Ipsi2_2 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 2 |
| SA323156 | VolpedoTissue_Ipsi2_3 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 2, technical replicate 3 |
| SA323157 | VolpedoTissue_Ipsi3_1 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 1 |
| SA323158 | VolpedoTissue_Ipsi3_2 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 2 |
| SA323159 | VolpedoTissue_Ipsi3_3 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 3, technical replicate 3 |
| SA323160 | VolpedoTissue_Ipsi4_1 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 1 |
| SA323161 | VolpedoTissue_Ipsi4_2 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 2 |
| SA323162 | VolpedoTissue_Ipsi4_3 | Tissue from the ipsilateral ear of an L. mexicana-infected mouse. Biological replicate 4, technical replicate 3 |
| Showing results 1 to 66 of 66 |
Collection:
| Collection ID: | CO003080 |
| Collection Summary: | For in vivo studies, the ears were collected, snap frozen, and processed for mass spectrometry analysis. For in vitro experiments, the culture supernatant was collected and cell debris was removed by centrifugation. The attached cells were be scraped, washed with PBS and quenched with 80% methanol. Then they were snap-frozen, centrifuged, and lyophilized |
| Collection Protocol Filename: | Study_Methods-Volpedo_et_al.pdf |
| Sample Type: | Biopsy |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR003096 |
| Treatment Summary: | Experimental mice were infected intradermally in the ear with 1 million L. mexicana promastigotes. Naive mice received PBS injection. RAW 264.7 macrophages and BMDMs were plated in a 24-well plate at a density of 0.5x106 per well and infected overnight with stationary phase L. mexicana promastigotes at a ratio of 10:1 (parasite to macrophages). The controls were treated with media alone. Then, the extracellular parasites were removed by washing with PBS and new media was applied. After a 24hrs incubation, the supernatant and cell pellet were collected and processed for mass spectrometry. |
| Treatment Protocol Filename: | Study_Design_and_Table-Volpedo_et_al.pdf |
Sample Preparation:
| Sampleprep ID: | SP003093 |
| Sampleprep Summary: | For in vitro experiments, the culture supernatant was collected and cell debris was removed by centrifugation according to SOP 5 of the Laboratory Guide for Metabolomics Experiments. The attached cells were be scraped, washed with PBS and quenched with 80% methanol. Then they were snap-frozen, centrifuged, and lyophilized according to SOP 4 of the Laboratory Guide for Metabolomics Experiments. For in vivo studies, the ears were collected, snap frozen, and processed for mass spectrometry analysis according to SOP 7 of the Laboratory Guide for Metabolomics Experiments. Samples were then incubated with 500 uL of 100% MeOH and sonicated. The tissue was weighed and homogenized at 40 mg/mL of 50% MeOH solution for 3 cycles in a Precellys homogenizer. The supernatant was collected, dried down, and reconstituted in ½ of the original volume in 5 % MeOH with 0.1 % formic acid. For metabolite candidates found via untargeted analysis, pure standards purchased from Sigma-Aldrich were diluted in 100 % MeOH stocks to 10 ug/mL, dried down, and reconstituted in 5 % MeOH with 0.1 % formic acid and run in the same conditions described below to match features for identification. Untargeted analysis was performed on a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (MS) with HPLC separation on a Poroshell 120 SB-C18 (2 x 100 mm, 2.7 µm particle size) with a Thermo Scientific Ultimate WPS 3000 UHPLC system. The gradient consisted of solvent A, H2O with 0.1% Formic acid, and solvent B 100% acetonitrile at a 200 µL/min flow rate with an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding at 95% B for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B until min 30. For each sample, 5 µL was injected for each sample with ionization in the MS on a HESI electrospray ionization source using a capillary voltage of 4.5 kV in positive and 4.0 kV in negative mode at 320 °C capillary temperature and 100 °C probe temperature. Gas settings were set to 15 for sheath gas and 5 auxiliary gas while the S-Lens was set to 50 V. The top 5 ions were selected for data dependent analysis with a 10 second exclusion window, with a mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and 17,5000 for MSMS scans and fragmentation normalized collision energies of 30 V. For feature selection in the untargeted results analysis, including database comparison and statistical processing, samples were analyzed in Progenesis QI and the pooled sample runs were selected for feature alignment. Using the pooled QC samples, features with above 30% CV and max abundances below 5000 intensity were filtered out and Anova p-value scores between the groups were calculated with a cutoff of < 0.05. With database matching using the Human Metabolome Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were tentatively identified as potential metabolites. Metabolites were only annotated with MSMS fragmentation matching scores above 20 % with Progenesis Metascope. |
| Sampleprep Protocol Filename: | Study_Methods-Volpedo_et_al.pdf |
Combined analysis:
| Analysis ID | AN004883 | AN004884 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Thermo Scientific Ultimate WPS 3000 UHPLC system | Thermo Scientific Ultimate WPS 3000 UHPLC system |
| Column | Agilent Infinity Poroshell 120 SB-C18 (100 x 2 mm,2.7um) | Agilent Infinity Poroshell 120 SB-C18 (100 x 2 mm,2.7um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Plus Orbitrap | Thermo Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | ppm | ppm |
Chromatography:
| Chromatography ID: | CH003685 |
| Chromatography Summary: | Untargeted analysis was performed on a Thermo Scientific Q-Exactive Plus Orbitrap mass spectrometer (MS) with HPLC separation on a Poroshell 120 SB-C18 (2 x 100 mm, 2.7 µm particle size) with a Thermo Scientific Ultimate WPS 3000 UHPLC system. The gradient consisted of solvent A, H2O with 0.1% Formic acid, and solvent B 100% acetonitrile at a 200 µL/min flow rate with an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding at 95% B for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B until min 30. For each sample, 5 µL was injected for each sample with ionization in the MS on a HESI electrospray ionization source using a capillary voltage of 4.5 kV in positive and 4.0 kV in negative mode at 320 °C capillary temperature and 100 °C probe temperature. Gas settings were set to 15 for sheath gas and 5 auxiliary gas while the S-Lens was set to 50 V. |
| Methods Filename: | Study_Methods-Volpedo_et_al.pdf |
| Instrument Name: | Thermo Scientific Ultimate WPS 3000 UHPLC system |
| Column Name: | Agilent Infinity Poroshell 120 SB-C18 (100 x 2 mm,2.7um) |
| Column Temperature: | 320 °C capillary temperature and 100 °C probe temperature |
| Flow Gradient: | an initial 2% solvent B with a linear ramp to 95% B at 15 min, holding at 95% B for 1 minutes, and back to 5% B from 17 min and equilibration of 5% B until min 30 |
| Flow Rate: | 200 µL/min flow rate |
| Solvent A: | H2O with 0.1% Formic acid |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS004627 |
| Analysis ID: | AN004883 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See Study Methods for full procedures. After LC-MS run, the top 5 ions were selected for data dependent analysis with a 10 second exclusion window, with a mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and 17,5000 for MSMS scans and fragmentation normalized collision energies of 30 V. For feature selection in the untargeted results analysis, including database comparison and statistical processing, samples were analyzed in Progenesis QI and the pooled sample runs were selected for feature alignment. Using the pooled QC samples, features with above 30% CV and max abundances below 5000 intensity were filtered out and Anova p-value scores between the groups were calculated with a cutoff of < 0.05. With database matching using the Human Metabolome Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were tentatively identified as potential metabolites. Metabolites were only annotated with MSMS fragmentation matching scores above 20 % with Progenesis Metascope. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | Study_Methods-Volpedo_et_al.pdf |
| MS ID: | MS004628 |
| Analysis ID: | AN004884 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | See Study Methods for full procedures. After LC-MS run, the top 5 ions were selected for data dependent analysis with a 10 second exclusion window, with a mass range of 80-1200 m/z and a resolution of 70,000 for MS scans and 17,5000 for MSMS scans and fragmentation normalized collision energies of 30 V. For feature selection in the untargeted results analysis, including database comparison and statistical processing, samples were analyzed in Progenesis QI and the pooled sample runs were selected for feature alignment. Using the pooled QC samples, features with above 30% CV and max abundances below 5000 intensity were filtered out and Anova p-value scores between the groups were calculated with a cutoff of < 0.05. With database matching using the Human Metabolome Database, selecting for adducts M+H, M+Na, M+2H, and 2M+H for positive mode and M-H, M+Cl, M-2H, and 2M-H and less than 10 ppm mass error, unique features were tentatively identified as potential metabolites. Metabolites were only annotated with MSMS fragmentation matching scores above 20 % with Progenesis Metascope. |
| Ion Mode: | NEGATIVE |
| Analysis Protocol File: | Study_Methods-Volpedo_et_al.pdf |
