Summary of Study ST002328

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 PR001492. The data can be accessed directly via it's Project DOI: 10.21228/M8VX2Q This work is supported by NIH grant, U2C- DK119886.

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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 IDST002328
Study TitleMetabolome and transcriptome analysis of oral mucosa of HIV+ patients reveal a role for polyamine metabolic pathway in T cell dysfunction
Study SummaryMetabolic changes of immune cells contribute to both physiological and pathophysiological outcomes of immune reactions. How viruses alter the metabolic states of mucosal T cells and the precise mechanisms underlying the persisting immune dysfunction during chronic viral infections are key questions that have not been fully addressed. Here, by comparing transcriptome and salivary metabolome profiles of the uninfected individuals and people living with HIV (PLWH) on treatment, we found a role of polyamine metabolism in immune perturbations of the oral mucosa of HIV+ patients. Flow cytometry analysis confirmed the higher expression of ornithine decarboxylase (ODC-1) and eukaryotic translation initiation factor 5A (EIF5A), the polyamine metabolism intermediates in CD4+ T cells in PLWH. Mechanistic studies using an in vitro human tonsil organoid infection model revealed that HIV infection of activated T cells also resulted in increased polyamine synthesis, which was dependent on the activities of caspase-1, IL-1β, and ODC-1. HIV-1 also led to elevated dysfunctional regulatory T cells (TregDys) /Thelper 17 (Th17) cell ratios as well as heightened expression of ODC-1, EIF5A, and hypusinated EIF5A. Blockade of caspase-1, ODC-1, and EIF5A hypusination and not HIF-1⍺ or NLRP3 reversed the frequency of TregDys showing the direct impact of polyamine pathway in Treg dysfunction during HIV-1 infection. The addition of exogenous polyamines increased TregDys percentages independent of HIV-1 infection in vitro. Finally, oral mucosal TregDys/Th17 ratios and CD4 hyperactivation positively correlated with the increases in salivary putrescine levels, which were found to be elevated in the saliva of PLWH. Thus, by revealing the role of aberrantly increased polyamine synthesis during HIV infection, our study unveils a new mechanism by which chronic viral infections could drive distinct T cell effector programs and Treg dysfunction.
Institute
Case Western Reserve University
DepartmentBiological Sciences
LaboratoryPushpa Pandiyan
Last NamePandiyan
First NamePushpa
AddressDepartment of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, Ohio, 44106
Emailpxp226@case.edu
Phone216-269-2939
Submit Date2022-08-29
Num Groups2
Total Subjects66
Num Males41
Num Females25
PublicationsUnder revision
Raw Data AvailableYes
Raw Data File Type(s)raw(Waters)
Analysis Type DetailLC-MS
Release Date2022-12-01
Release Version1
Pushpa Pandiyan Pushpa Pandiyan
https://dx.doi.org/10.21228/M8VX2Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001492
Project DOI:doi: 10.21228/M8VX2Q
Project Title:Metabolome and transcriptome analysis of oral mucosa of HIV+ patients reveal a role for polyamine metabolic pathway in T cell dysfunction
Project Type:Clinical research
Project Summary:Metabolic changes of immune cells contribute to both physiological and pathophysiological outcomes of immune reactions. How viruses alter the metabolic states of mucosal T cells and the precise mechanisms underlying the persisting immune dysfunction during chronic viral infections are key questions that have not been fully addressed. Here, by comparing transcriptome and salivary metabolome profiles of the uninfected individuals and people living with HIV (PLWH) on treatment, we found a role of polyamine metabolism in immune perturbations of the oral mucosa of HIV+ patients. Flow cytometry analysis confirmed the higher expression of ornithine decarboxylase (ODC-1) and eukaryotic translation initiation factor 5A (EIF5A), the polyamine metabolism intermediates in CD4+ T cells in PLWH. Mechanistic studies using an in vitro human tonsil organoid infection model revealed that HIV infection of activated T cells also resulted in increased polyamine synthesis, which was dependent on the activities of caspase-1, IL-1β, and ODC-1. HIV-1 also led to elevated dysfunctional regulatory T cells (TregDys) /Thelper 17 (Th17) cell ratios as well as heightened expression of ODC-1, EIF5A, and hypusinated EIF5A. Blockade of caspase-1, ODC-1, and EIF5A hypusination and not HIF-1⍺ or NLRP3 reversed the frequency of TregDys showing the direct impact of polyamine pathway in Treg dysfunction during HIV-1 infection. The addition of exogenous polyamines increased TregDys percentages independent of HIV-1 infection in vitro. Finally, oral mucosal TregDys/Th17 ratios and CD4 hyperactivation positively correlated with the increases in salivary putrescine levels, which were found to be elevated in the saliva of PLWH. Thus, by revealing the role of aberrantly increased polyamine synthesis during HIV infection, our study unveils a new mechanism by which chronic viral infections could drive distinct T cell effector programs and Treg dysfunction.
Institute:Case Western Reserve University
Department:Biological Sciences
Laboratory:Pushpa Pandiyan
Last Name:Pandiyan
First Name:Pushpa
Address:Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, Ohio, 44106
Email:pxp226@case.edu
Phone:216-368-2939
Funding Source:NIH/NIDCR RO1DE026923
Publications:Under revision
Contributors:Mahalingam SS, Jayaraman S, Bhaskaran N, Schneider E, Faddoul F, Paes da Silva A, Lederman MM, Asaad R, Adkins-Travis K, Shriver LP, and Pandiyan P

Subject:

Subject ID:SU002415
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:N/A
Age Or Age Range:19-75
Weight Or Weight Range:N/A
Height Or Height Range:N/A
Gender:Male and female

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA229509A3gControl
SA229510A3hControl
SA229511A3fControl
SA229512A3eControl
SA229513A3dControl
SA229514A3iControl
SA229515A3jControl
SA229516A1aControl
SA229517A3nControl
SA229518A3mControl
SA229519A3lControl
SA229520A3cControl
SA229521A3kControl
SA229522A1dControl
SA229523A1fControl
SA229524A1cControl
SA229525A3bControl
SA229526A1bControl
SA229527A2aControl
SA229528A1eControl
SA229529A3aControl
SA229530A2bControl
SA229531A2fControl
SA229532A2eControl
SA229533A2dControl
SA229534A2cControl
SA229535B3pPLWH
SA229536B3rPLWH
SA229537B3oPLWH
SA229538B3qPLWH
SA229539B3mPLWH
SA229540B3kPLWH
SA229541B3sPLWH
SA229542B3lPLWH
SA229543B3nPLWH
SA229544B3zPLWH
SA229545B3aaPLWH
SA229546B3bbPLWH
SA229547B3jPLWH
SA229548B3yPLWH
SA229549B3xPLWH
SA229550B3uPLWH
SA229551B3vPLWH
SA229552B3wPLWH
SA229553B3tPLWH
SA229554B2bPLWH
SA229555B1fPLWH
SA229556B2aPLWH
SA229557B2cPLWH
SA229558B2dPLWH
SA229559B1ePLWH
SA229560B1dPLWH
SA229561B1aPLWH
SA229562B1bPLWH
SA229563B1cPLWH
SA229564B2ePLWH
SA229565B2fPLWH
SA229566B3fPLWH
SA229567B3gPLWH
SA229568B3hPLWH
SA229569B3ePLWH
SA229570B3dPLWH
SA229571B3aPLWH
SA229572B3bPLWH
SA229573B3cPLWH
SA229574B3iPLWH
Showing results 1 to 66 of 66

Collection:

Collection ID:CO002408
Collection Summary:Samples were collected in 50 mL sterile centrifuge tubes and immediately stored at −80°C. 100 μl frozen aliquots of human saliva samples were processed for metabolite extraction liquid chromatography-mass spectrometry (LC-MS) analysis commercially by Creative Proteomics.
Collection Protocol Filename:Collection_protocol.pdf
Sample Type:Saliva
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002427
Treatment Summary:Samples were collected from both the healthy subjects (Control) and subjects living with HIV+ under anti-retroviral therapy (PLWH) in 50 ml sterile centrifuge tubes and immediately stored at −80°C. 100 μl frozen aliquots of human saliva samples were processed for metabolite extraction liquid chromatography-mass spectrometry (LC-MS) analysis commercially by Creative Proteomics.
Treatment Protocol Filename:Treatment_protocol.pdf

Sample Preparation:

Sampleprep ID:SP002421
Sampleprep Summary:100 μl of saliva samples were thawed, transferred to new tubes, extracted with 200 μl of 80% methanol, and vortexed for 30 seconds. Then the samples were kept at -40 °C for 1 hour, vortexed for 30 s, and centrifuged at 12000 rpm, 4 °C for 15 minutes. Finally, 200 μl of supernatant and 5 μl of DL-o-Chlorophenylalanine (1 mg/ml) were transferred to the vial for LC-MS analysis. Quality control (QC) samples were used to evaluate the methodology. The same amount of extract was obtained from each sample and mixed with QC samples. The QC sample was prepared using the same sample preparation procedure. Instrumental setup Separation was performed by Ultimate 3000LC combined with Q Exactive MS (Thermo) and screened with ESI-MS (targeted MS/MS mode). The LC system is comprised of an ACQUITY UPLC HSS T3 (100 × 2.1mm 1.8 μm) with Ultimate 3000LC. The mobile phase was composed of solvent A (0.05% formic acid-water) and solvent B (acetonitrile) with a gradient elution (0-1.0 min, 95% A; 1.0-12.0 min,95%-5% A; 12.0-13.5 min,5% A; 13.5-13.6 min, 5%-95% A; 13.6-16 min, 95% A). The flow rate of the mobile phase was 0.3 ml/min. The column temperature was maintained at 40 °C, and the sample manager temperature is set at 4 °C. Mass spectrometry parameters in ESI+ and ESI- mode are listed as follows: ESI+: Heater Temp 300 °C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 kV; Capillary Temp, 350 °C; S-Lens RF Level, 30%. ESI-: Heater Temp 300 °C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 kV; Capillary Temp,350 °C; S-Lens RF Level, 60%. Bioinformatic data analysis included multivariate statistical analysis, single variable analysis, cluster analysis, and correlation network of differential metabolites. Statistically significant metabolites (FC >1.5) were integrated with differentially expressed genes obtained from the RNAseq data and the combined data was visualized with Metaboanalyst (https://www.metaboanalyst.ca/) and Cytoscape v3.8 via Metscape v3.1 plugin (https://cytoscape.org/).
Sampleprep Protocol Filename:Sample_preparation.pdf
Processing Storage Conditions:Described in summary
Extract Storage:Described in summary

Combined analysis:

Analysis ID AN003797 AN003798
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Thermo Dionex Ultimate 3000 Thermo Dionex Ultimate 3000
Column Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um) Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode POSITIVE NEGATIVE
Units area area

Chromatography:

Chromatography ID:CH002810
Methods Filename:Chromatography_meta_data_queriesCP.pdf
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:40
Flow Gradient:(0-1.0 min, 95% A; 1.0-12.0 min,95%-5% A; 12.0-13.5 min,5% A; 13.5-13.6 min, 5%- 629 95% A; 13.6-16 min, 95% A
Flow Rate:.3 ml/min
Injection Temperature:4
Solvent A:100% water; 0.05% formic acid
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS003539
Analysis ID:AN003797
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolomics was performed by Creative Proteomics. Gingival cells enriched in immune cells were prepared by removing epithelial cells based on gradient centrifugation. HOIL samples from three control individuals were pooled and compared with three independent HIV+ individuals. Separation was performed by Ultimate 3000LC combined with Q Exactive MS (Thermo) and screened with ESI-MS (targeted MS/MS mode). The software we used for metabolites identification is Compound Discoverer 3.1 SP1. For metabolites identification, the raw data are acquired and aligned using the Compound Discover based on the m/z value and the retention time of the ion signals. CD software offers a fully integrated suite of advanced software tools for known-parent and unknown data processing and interpretation. It is used for the initial metabolites identification. We also use mzMine2 software (with parameters of m/z tolerance=0.1 m/z ) for the online database search based on the MS1 and MS2 spectra acquired. The database used is HMDB.
Ion Mode:POSITIVE
Analysis Protocol File:MS_Metadata.docx
  
MS ID:MS003540
Analysis ID:AN003798
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolomics was performed by Creative Proteomics. Gingival cells enriched in immune cells were prepared by removing epithelial cells based on gradient centrifugation. HOIL samples from three control individuals were pooled and compared with three independent HIV+ individuals. Separation was performed by Ultimate 3000LC combined with Q Exactive MS (Thermo) and screened with ESI-MS (targeted MS/MS mode). The software we used for metabolites identification is Compound Discoverer 3.1 SP1. For metabolites identification, the raw data are acquired and aligned using the Compound Discover based on the m/z value and the retention time of the ion signals. CD software offers a fully integrated suite of advanced software tools for known-parent and unknown data processing and interpretation. It is used for the initial metabolites identification. We also use mzMine2 software (with parameters of m/z tolerance=0.1 m/z ) for the online database search based on the MS1 and MS2 spectra acquired. The database used is HMDB.
Ion Mode:NEGATIVE
Analysis Protocol File:MS_Metadata.docx
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