Summary of Study ST001922

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 PR001213. The data can be accessed directly via it's Project DOI: 10.21228/M8X70P This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST001922
Study Title	Sublytic membrane attack complex drives glycolysis and mitochondrial dysfunction with inflammatory consequences in human monocyte-derived macrophages
Study Summary	The terminal stage in the complement activation pathways, the membrane attack complex (MAC), is upregulated in diabetic and rheumatoid arthritis patients, contributing pathologically by increasing inflammation. Previous research has highlighted that a sublytic dose of MAC can initiate NLRP3 inflammasome activation via calcium influx and loss of mitochondrial membrane potential. Here, we show that sublytic concentrations of MAC mediate a previously undescribed perturbation in cellular energy metabolism in human monocyte-derived macrophages, by phenotypic skewing towards glycolysis and upregulation of glycolysis-promoting genes. Sublytic MAC concentrations drive mitochondrial dysfunction, characterised by a fragmented mitochondrial morphology, loss of maximal respiratory response, depleted mitochondrial membrane potential as well as increased mitochondrial reactive oxygen species production. The consequences of these alterations in glycolytic metabolism and mitochondrial dysfunction lead to NLRP3 inflammasome activation, driving gasdermin D formation and IL-18 release. This novel link between sublytic MAC and immunometabolism, with direct consequences for downstream inflammatory processes, is important for development of novel therapeutics for areas where MAC may mediate disease.
Institute	GSK
Department	Discovery Analytical
Laboratory	MST-MedDesign
Last Name	Kozole
First Name	Joseph
Address	1250 Collegeville Ave, Upper Providence, PA, US
Email	joseph.x.kozole@gsk.com
Phone	8144410679
Submit Date	2021-09-23
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2021-10-18
Release Version	1

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Sample Preparation:

Sampleprep ID:	SP002006
Sampleprep Summary:	hMDMs at 1 million cells/well in 24-well plates were sensitized to complement as usual and treated with a sublytic dose of NHS or left untreated for 4 hours. Following treatment, supernatants were removed and the hMDMs rinsed with fresh assay media (RPMI-1640 with 2mM L-Glutamine). For metabolite exaction, 75% 9:1 MeOH:CHCl3 was added directly to wells containing frozen cells (-80 °C). Cells from four technical replicates for each donor and sample type (NHS treated and untreated) were scraped from their respective wells, combined into Covaris adaptive focused acoustic tubes (for a total of 4 million cells per sample), and disrupted using a 2 minute lysis method on a Covaris S220 Focused Ulrasonicator (Peak Power - 200, Duty Factor - 10, Cycle/Burst - 200). Lysed samples were centrifuged at 5000 x g for 15 minutes at room temperature. Supernatant was split into two equal fractions and set on a Speedvac concentrator to dryness. Samples were reconstituted in either 3:1 MeCN:H2O or H2O+0.1% formic acid for analysis by LC-MS/MS with hydrophilic interaction (HILIC) or reverse phase chromatography respectively.

Sampleprep ID:

SP002006

Sampleprep Summary:

hMDMs at 1 million cells/well in 24-well plates were sensitized to complement as usual and treated with a sublytic dose of NHS or left untreated for 4 hours. Following treatment, supernatants were removed and the hMDMs rinsed with fresh assay media (RPMI-1640 with 2mM L-Glutamine). For metabolite exaction, 75% 9:1 MeOH:CHCl3 was added directly to wells containing frozen cells (-80 °C). Cells from four technical replicates for each donor and sample type (NHS treated and untreated) were scraped from their respective wells, combined into Covaris adaptive focused acoustic tubes (for a total of 4 million cells per sample), and disrupted using a 2 minute lysis method on a Covaris S220 Focused Ulrasonicator (Peak Power - 200, Duty Factor - 10, Cycle/Burst - 200). Lysed samples were centrifuged at 5000 x g for 15 minutes at room temperature. Supernatant was split into two equal fractions and set on a Speedvac concentrator to dryness. Samples were reconstituted in either 3:1 MeCN:H2O or H2O+0.1% formic acid for analysis by LC-MS/MS with hydrophilic interaction (HILIC) or reverse phase chromatography respectively.