Summary of Study ST003290

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 PR002042. The data can be accessed directly via it's Project DOI: 10.21228/M8NV6P 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	ST003290
Study Title	High expression of oleoyl-ACP-hydrolase underpins life-threatening respiratory viral diseases
Study Summary	Although respiratory viral infections cause significant morbidity and mortality, it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning hypercytokinemia and fatal disease. Our transcriptomics studies identified differential expression of 10 early host genes, defined by 16 probe sets, between patients who recovered and died. Seven probe sets were specific for the same host gene encoding for a key enzyme mediating free fatty acid production, oleoyl-ACP-hydrolase (OLAH). High OLAH levels in fatal A(H7N9) patients were detected early after hospital admission and persisted until patients died. Conversely, patients who recovered had minimal OLAH expression throughout their hospital stay. High OLAH levels were also detected in patients hospitalized for severe infections with seasonal influenza virus, SARS-CoV-2, respiratory syncytial virus (RSV) and for multisystem inflammatory syndrome in children (MIS-C), while the main catalytic product of OLAH, oleic acid, was increased in hospitalized compared to ambulatory patients. Among healthy individuals and those with mild infections, however, OLAH was minimally detected. To understand how OLAH drives disease severity, we generated olah deficient mice. In contrast to wild-type mice, lethal influenza infection of olah-/- mice led to survival, milder disease, and markedly reduced lung viral loads, tissue damage, infection-driven pulmonary innate cell infiltration and inflammatory milieu. This phenotype was associated with differential lipid droplet dynamics, and reduced viral infection and inflammatory responses in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza infection of macrophages and their inflammatory potential. Increased infectivity in the presence of olah was dependent on lipid droplet usage. Our findings define how expression of the key host enzyme, OLAH, drives life-threatening inflammation associated with respiratory viruses and propose OLAH as a potential early target for diagnosis and treatment of patients with severe disease.
Institute	Peter Doherty Institute for Infection and Immunity
Last Name	Chua
First Name	Brendon
Address	792 Elizabeth St, Melbourne VIC 3000
Email	bychua@unimelb.edu.au
Phone	+61383441130
Submit Date	2024-06-26
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-08-22
Release Version	1

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Project:

Project ID:	PR002042
Project DOI:	doi: 10.21228/M8NV6P
Project Title:	High expression of oleoyl-ACP-hydrolase underpins life-threatening respiratory viral diseases
Project Summary:	Although respiratory viral infections cause significant morbidity and mortality, it is unclear why some individuals succumb to severe disease. In patients hospitalized with avian A(H7N9) influenza, we investigated early drivers underpinning hypercytokinemia and fatal disease. Our transcriptomics studies identified differential expression of 10 early host genes, defined by 16 probe sets, between patients who recovered and died. Seven probe sets were specific for the same host gene encoding for a key enzyme mediating free fatty acid production, oleoyl-ACP-hydrolase (OLAH). High OLAH levels in fatal A(H7N9) patients were detected early after hospital admission and persisted until patients died. Conversely, patients who recovered had minimal OLAH expression throughout their hospital stay. High OLAH levels were also detected in patients hospitalized for severe infections with seasonal influenza virus, SARS-CoV-2, respiratory syncytial virus (RSV) and for multisystem inflammatory syndrome in children (MIS-C), while the main catalytic product of OLAH, oleic acid, was increased in hospitalized compared to ambulatory patients. Among healthy individuals and those with mild infections, however, OLAH was minimally detected. To understand how OLAH drives disease severity, we generated olah deficient mice. In contrast to wild-type mice, lethal influenza infection of olah-/- mice led to survival, milder disease, and markedly reduced lung viral loads, tissue damage, infection-driven pulmonary innate cell infiltration and inflammatory milieu. This phenotype was associated with differential lipid droplet dynamics, and reduced viral infection and inflammatory responses in macrophages. Supplementation of oleic acid, the main product of OLAH, increased influenza infection of macrophages and their inflammatory potential. Increased infectivity in the presence of olah was dependent on lipid droplet usage. Our findings define how expression of the key host enzyme, OLAH, drives life-threatening inflammation associated with respiratory viruses and propose OLAH as a potential early target for diagnosis and treatment of patients with severe disease.
Institute:	Peter Doherty Institute for Infection and Immunity
Last Name:	Chua
First Name:	Brendon
Address:	792 Elizabeth St, Melbourne VIC 3000
Email:	bychua@unimelb.edu.au
Phone:	+61383441130

Subject:

Subject ID:	SU003410
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

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

mb_sample_id	local_sample_id	Sample source	Mouse Strain	Infection status
SA356498	231201_BC-7661_blank_4	Extraction blank	Extraction blank	Extraction blank
SA356499	231201_BC-7661_blank_3	Extraction blank	Extraction blank	Extraction blank
SA356500	231201_BC-7661_blank_2	Extraction blank	Extraction blank	Extraction blank
SA356501	231201_BC-7661_blank_1	Extraction blank	Extraction blank	Extraction blank
SA356502	231201_BC-7661_20	Lung	C57BL/6 wildtype	Infected
SA356503	231201_BC-7661_19	Lung	C57BL/6 wildtype	Infected
SA356504	231201_BC-7661_18	Lung	C57BL/6 wildtype	Infected
SA356505	231201_BC-7661_17	Lung	C57BL/6 wildtype	Infected
SA356506	231201_BC-7661_16	Lung	C57BL/6 wildtype	Infected
SA356507	231201_BC-7661_14	Lung	C57BL/6 wildtype	Naïve
SA356508	231201_BC-7661_15	Lung	C57BL/6 wildtype	Naïve
SA356509	231201_BC-7661_13	Lung	C57BL/6 wildtype	Naïve
SA356510	231201_BC-7661_12	Lung	C57BL/6 wildtype	Naïve
SA356511	231201_BC-7661_11	Lung	C57BL/6 wildtype	Naïve
SA356512	231201_BC-7661_10	Lung	olah-/-	Infected
SA356513	231201_BC-7661_9	Lung	olah-/-	Infected
SA356514	231201_BC-7661_8	Lung	olah-/-	Infected
SA356515	231201_BC-7661_7	Lung	olah-/-	Infected
SA356516	231201_BC-7661_6	Lung	olah-/-	Infected
SA356517	231201_BC-7661_2	Lung	olah-/-	Naïve
SA356518	231201_BC-7661_5	Lung	olah-/-	Naïve
SA356519	231201_BC-7661_4	Lung	olah-/-	Naïve
SA356520	231201_BC-7661_3	Lung	olah-/-	Naïve
SA356521	231201_BC-7661_1	Lung	olah-/-	Naïve

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO003403
Collection Summary:	Whole lungs were harvested, weighed, minced and freeze dried. Prior to lipid extraction, freeze dried lungs were incubated with 200µl of ice-cold 60% methanol containing 0.01% (w/v) butylated hydroxytoluene (BHT) and incubated overnight at -20°C
Sample Type:	Lung

Treatment:

Treatment ID:	TR003419
Treatment Summary:	Mice (olah-/- or C57BL/6) were bred and maintained in the Biological Research Facility in the Department of Microbiology and Immunology at The University of Melbourne. Following weaning (~3 weeks old), mice were placed on a normal maintenance diet (Barastoc, Ridley Corporation, Australia). Animal experiments were conducted in accordance with the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes Guidelines and institutional regulations following approval (permit numbers 1714304, 1614073 and 20532) by The University of Melbourne Animal Ethics Committee. Influenza virus infection was performed under light anaesthesia with isofluorane and intranasal instillation (30µl) with 2x104 plaque forming units of A/HK/x31 (X31; H3N2). One day following infection, lungs were harvested, weighed and snap frozen in preparation for lipid extraction. Naïve mice were treated as above except that influenza virus infection was not performed.

Treatment ID:

TR003419

Treatment Summary:

Mice (olah-/- or C57BL/6) were bred and maintained in the Biological Research Facility in the Department of Microbiology and Immunology at The University of Melbourne. Following weaning (~3 weeks old), mice were placed on a normal maintenance diet (Barastoc, Ridley Corporation, Australia). Animal experiments were conducted in accordance with the Australian National Health and Medical Research Council Code of Practice for the Care and Use of Animals for Scientific Purposes Guidelines and institutional regulations following approval (permit numbers 1714304, 1614073 and 20532) by The University of Melbourne Animal Ethics Committee. Influenza virus infection was performed under light anaesthesia with isofluorane and intranasal instillation (30µl) with 2x104 plaque forming units of A/HK/x31 (X31; H3N2). One day following infection, lungs were harvested, weighed and snap frozen in preparation for lipid extraction. Naïve mice were treated as above except that influenza virus infection was not performed.

Sample Preparation:

Sampleprep ID:	SP003417
Sampleprep Summary:	Whole lungs were harvested, weighed, minced and freeze dried. Prior to lipid extraction, freeze dried lungs were incubated with 200µl of ice-cold 60% methanol containing 0.01% (w/v) butylated hydroxytoluene (BHT) and incubated overnight at -20°C. SPLASH® LIPIDOMIX® Mass Spec Standard (10µl; Cat. 330707, Avanti Polar Lipids, Birmingham, AL, USA) and deuterated saturated/monounsaturated fatty acid standards (10µl; Cayman Chemical, USA) were added to each sample and tissue homogenised with a Bullet Blender (Next Advance, Troy, NY, USA) and sonicated for 20 minutes. Monophasic lipid extraction was performed according to Lydic et al (Lydic et al., 2015, DOI: 10.1016/j.ymeth.2015.04.014) wherein 120 μl of water, 420 μl of methanol with 0.01% (w/v) BHT, and 270 μl of chloroform were added to all samples prior to thorough vortexing and incubated on a shaker at 1400 rpm for 30 mins. Following centrifugation, supernatants containing lipids were transferred to new tubes and the remaining pellets re-extracted by adding 100µl of water and 400µl of chloroform:methanol (1:2 v/v) containing 0.01% (w/v) BHT and incubation on a shaker at 1000 rpm for 15 mins. Supernatants from each extraction round were pooled, dried by evaporation under vacuum and freeze-dried. Lyophilised samples were then resuspended in chloroform:methanol (1:9 v/v) containing 0.01% BHT and centrifuged to remove precipitates.

Sampleprep ID:

SP003417

Sampleprep Summary:

Whole lungs were harvested, weighed, minced and freeze dried. Prior to lipid extraction, freeze dried lungs were incubated with 200µl of ice-cold 60% methanol containing 0.01% (w/v) butylated hydroxytoluene (BHT) and incubated overnight at -20°C. SPLASH® LIPIDOMIX® Mass Spec Standard (10µl; Cat. 330707, Avanti Polar Lipids, Birmingham, AL, USA) and deuterated saturated/monounsaturated fatty acid standards (10µl; Cayman Chemical, USA) were added to each sample and tissue homogenised with a Bullet Blender (Next Advance, Troy, NY, USA) and sonicated for 20 minutes. Monophasic lipid extraction was performed according to Lydic et al (Lydic et al., 2015, DOI: 10.1016/j.ymeth.2015.04.014) wherein 120 μl of water, 420 μl of methanol with 0.01% (w/v) BHT, and 270 μl of chloroform were added to all samples prior to thorough vortexing and incubated on a shaker at 1400 rpm for 30 mins. Following centrifugation, supernatants containing lipids were transferred to new tubes and the remaining pellets re-extracted by adding 100µl of water and 400µl of chloroform:methanol (1:2 v/v) containing 0.01% (w/v) BHT and incubation on a shaker at 1000 rpm for 15 mins. Supernatants from each extraction round were pooled, dried by evaporation under vacuum and freeze-dried. Lyophilised samples were then resuspended in chloroform:methanol (1:9 v/v) containing 0.01% BHT and centrifuged to remove precipitates.

Combined analysis:

Analysis ID	AN005388	AN005389
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	Waters ACQUITY UPLC HSS T3 (150 x 1mm,1.8um)	Waters ACQUITY UPLC HSS T3 (150 x 1mm,1.8um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Fusion Tribrid Orbitrap	Thermo Fusion Tribrid Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	pmol/mg lung tissue	pmol/mg lung tissue

Chromatography:

Chromatography ID:	CH004085
Chromatography Summary:	Solvent A: 6/4 (v/v) acetonitrile/water with 5 uM medronic acid and 10 mM ammonium acetate; Solvent B: 9/1 (v/v) isopropanol/acetonitrile with 10 mM ammonium acetate
Instrument Name:	Thermo Vanquish
Column Name:	Waters ACQUITY UPLC HSS T3 (150 x 1mm,1.8um)
Column Temperature:	50
Flow Gradient:	[Time(min), %B solvent]: [0,3], [5,3], [10,70], [26,99], [29,99], [29.1,3], [33,3]
Flow Rate:	60 uL/min
Solvent A:	60% acetonitrile/40% water; 5 uM medronic acid; 10 mM ammonium acetate
Solvent B:	90% isopropyl alcohol/10% acetonitrile; 10 mM ammonium acetate
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005115
Analysis ID:	AN005388
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Top speed data-dependent scan with a cycle time of 1s was used. Within each cycle, a full-scan MS-spectra were acquired firstly in the Orbitrap at a mass resolving power of 120,000 across an m/z range of 300–2000 using quadrupole isolation, followed by higher-energy collisional dissociation (HCD)-MS/MS at a mass resolving power of 15,000 (at m/z 200) and a normalized collision energy (NCE) of 27% at positive mode and 30% in negative mode with an m/z isolation window of 1.
Ion Mode:	POSITIVE

MS ID:	MS005116
Analysis ID:	AN005389
Instrument Name:	Thermo Fusion Tribrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Top speed data-dependent scan with a cycle time of 1s was used. Within each cycle, a full-scan MS-spectra were acquired firstly in the Orbitrap at a mass resolving power of 120,000 across an m/z range of 300–2000 using quadrupole isolation, followed by higher-energy collisional dissociation (HCD)-MS/MS at a mass resolving power of 15,000 (at m/z 200) and a normalized collision energy (NCE) of 27% at positive mode and 30% in negative mode with an m/z isolation window of 1.
Ion Mode:	NEGATIVE