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Data from: Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice

Cite this dataset

Favor, Olivia et al. (2022). Data from: Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice [Dataset]. Dryad. https://doi.org/10.5061/dryad.h44j0zppx

Abstract

Lipopolysaccharide (LPS)-accelerated autoimmune glomerulonephritis (GN) in lupus-prone NZBWF1 mice is a preclinical model that is potentially applicable for investigating lipidome-modulating interventions. LPS can be expressed as one of two chemotypes: smooth LPS (S-LPS) and rough LPS (R-LPS) which is devoid of O-antigen polysaccharide sidechain. Since these chemotypes differentially affect TLR4-mediated immune cell responses, these differences may influence GN induction. Therefore, we initially compared the effects of subchronic i.p. injection for 5 wk with 1) Salmonella S-LPS, 2) Salmonella R-LPS, or 3) saline vehicle (VEH) (Study 1) in female NZBWF1 mice. R-LPS induced robust elevations in blood urea nitrogen, proteinuria, and hematuria that were not evident in VEH- or S-LPS-treated mice. Histopathologic examination of R-LPS-treated mice one week after final injection further revealed more robust hypertrophy, hyperplasia, thickened membranes, lymphocytic accumulation containing B and T cells, and glomerular IgG deposition consistent with GN but not in VEH- or S-LPS-treated groups. R-LPS but not S-LPS induced spleen enlargement with lymphoid hyperplasia as well as modest inflammatory cell recruitment in the liver. We next employed our optimized R-LPS model to discern the impact of two lipidome-modulating interventions, omega-3 polyunsaturated fatty acid (PUFA) supplementation and soluble epoxide hydrolase (sEH) inhibition, on GN (Study 2). Specifically, the effects of consuming the omega-3 PUFA docosahexaenoic acid (DHA) (10 g/kg diet) and/or the sEH inhibitor TPPU (22.5 mg/kg diet) on R-LPS triggering were compared. Resultant blood fatty acid profiles and epoxy fatty acid concentrations reflected the anticipated DHA- and TPPU-mediated lipidome changes. The relative rank order of R-LPS-induced GN severity among groups fed experimental diets based on proteinuria, hematuria, histopathologic scoring, and glomerular IgG deposition was: VEH/CON < R-LPS/DHA ≈ R-LPS/TPPU <<< R-LPS/ TPPU+DHA ≈ R-LPS/CON. These interventions had modest to negligible effects on R-LPS-induced splenomegaly, plasma antibody responses, liver inflammation, and inflammation-associated kidney gene expression. Collectively, our results show for the first time that absence of O-antigenic polysaccharide in R-LPS is critical to accelerated GN in lupus-prone mice. Furthermore, intervention by lipidome modulation through DHA feeding or sEH inhibition suppressed R-LPS-induced GN; however, these ameliorative effects were greatly diminished upon combining the treatments.

Methods

LC-MS/MS Quantitation of Plasma Oxylipins

Waters Oasis-HLB cartridges (part no. #WAT094226, lot no. #176A30323A) were used for sample preparation and clean-up purposes. Solid-phase extraction (SPE) cartridges were prepared for solid phase extraction by washing once with 2 ml of ethyl acetate, twice with 2 ml of methanol, and twice with 2 ml of 95:5 (v/v) water/methanol + 0.1% (v/v) acetic acid. Plasma was then loaded onto the cartridges, and samples were spiked with 10 μl of deuterated internal standard solution (16 nM BGB2-d4, 10 nM LTB4-d4, 16 nM 8,9-DiHETrE-d11, 16 nM 9-HODE-d4, 20 nM 15(S)-HETE-d8, 40 nM 5(S)-HETE-d8, 40 nM 8,9-EpETrE-d11) and 10 μl of antioxidant cocktail (0.2 mg/ml butylated hydroxytoluene, 0.2 mg/ml triphenylphosphine, 0.6 mg/ml EDTA). After loading samples, cartridges were washed with 1.5 ml of 95:5 (v/v) water/methanol + 0.1% (v/v) acetic acid. The SPE cartridges were dried with a low vacuum for about 20 minutes to pull out the water and other unwanted residues from the cartridge. SPE cartridges were eluted with 0.5 ml of methanol followed by 1 ml of ethyl acetate into 2 ml Eppendorf tubes containing 6 μl of 30% (v/v) glycerol in methanol as a trap solution. The eluents were concentrated under a high vacuum. The residues were reconstituted in 100 μl of 75% ethanol (v/v) containing 10 nM 12-[[(cyclohexylamino)carbonyl]amino]-dodecanoic acid (CUDA) as an internal standard. The samples were then vortexed for 5 min followed by filtration through a 0.45 μm filter. The filtrates were then transferred to LC-MS/MS vials for analysis.

A XBridge BEH C18 2.1x150 mm, 5 µm, HPLC column, (ser. #01723829118314) was used for ultra-performance liquid chromatography (UPLC). The column was connected to a Waters TQ-XS tandem quadrupole UPLC/MS/MS instrument equipped with a Waters Acquity SDS pump and Waters Acquity CM detector (Milford, MA). Mobile phase A comprised of 0.1% (v/v) acetic acid in water. Mobile phase B consisted of 84:16 (v/v) acetonitrile/methanol + 0.1% glacial acetic acid. Gradient elution was performed at a flow rate of 250 μl/min. Chromatography was optimized to separate all analytes in 20 min (Table 1). The Waters Acquity FTN autosampler (Milford, MA) was held at 10 °C during sample injection.

Electrospray was the ionization source for negative multiple reaction monitoring (MRM) modes. To achieve the best selectivity and sensitivity, each analyte standard was infused into the mass spectrometer, and MRM transitions and source parameters were optimized for that analyte. For each experimental sample, Waters MassLynx™ MS software v4 (Milford, MA) was used to quantify analyte area, internal standard (IS) area, raw concentration (in nM), and signal-to-noise (S/N) ratio based on an 8spots-calibration linear standard curve. Dilution factors were calculated for each sample by dividing the original sample volume (in µl) by 100 µl. Normalized analyte concentrations in each sample were then quantified by dividing raw analyte concentrations by the sample’s corresponding dilution factor.

High-Throughput Autoantibody Profiling

IgG and IgM autoantibodies (AAbs) were profiled in plasma by OmicsArray™ Systemic Autoimmune-associated Antigen Array (Genecopoiea Inc., Rockville, MD; cat. #PA001). All plasma samples within experimental groups were pooled prior to analysis. Antigen microarray plates coated with 120 purified antigens and 8 controls were incubated with experimental samples and then co-stained with Cy3-conjugated anti-mouse IgG (1:2000, Jackson ImmunoResearch Laboratories, PA) and Cy5-conjugated anti-mouse IgM (1:2000, Jackson ImmunoResearch Laboratories). Fluorescent signals were detected using a GenePix® 4400A microarray scanner (Molecular Devices, San Jose, CA), and GenePix® 7.0 software (Molecular Devices) was used to convert fluorescent signals to signal intensity values. Final values for all AAbs were converted into antibody scores (Ab-scores) based on normalized signal intensity (NSI) values and signal-to-noise ratio (SNR) using the following formula: Ab-score = log2(NSI x SNR + 1)

Kidney Gene Expression

Total RNA from kidneys was extracted using TissueLyser II (Qiagen, Germantown, MD) and a RNeasy Mini Kit (Qiagen; cat. #74104) according to the manufacturer’s instructions. Isolated RNA was reconstituted in RNase-free water and quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). RNA was reverse transcribed at a concentration of 100 ng/µl using a High-Capacity cDNA Reverse Transcriptase Kit (Thermo Fisher Scientific, Waltham, MA). Taqman assays were run with technical triplicates using a Smart Chip Real-Time PCR System at the MSU Genomics Core to assess interleukin (Il1a, Il1b, Il2, Il6, Il17a, Il18), chemokine (Ccl2, Ccl7, Ccl12, Cxcl9, Cxcl10, Cxcl13), inflammation/autoimmunity (C1qa, C3, Casp1, Casp4, Icam1, Ifng, Lbp, Nfkb1, Nlrp3, Nos2, Pparg, Tlr4, Tlr9, Tnfa, Tnfsf13b), type I interferon (IFN)-related (Ifi44, Irf7, Isg15, Nlrc5, Oas2), eicosanoid-related (Alox15, Cyp2c44, Cyp2j6, Cyp2j9, Cyp2j11, Ephx1, Ephx2, Pla2g4c, Ptgs2), kidney injury (Ankrd1, Cd14, Havcr1, Tgfbr1), oxidative stress-related (Hmox, Ncf1, Nqo1, Sod2), and housekeeping (Actb, Gusb) gene expression. Raw Ct values were converted to ΔCt values for each gene by subtracting the average Ct of the housekeeping genes from the Ct of the specified gene, and ΔΔCt values for each gene were calculated relative to the VEH/CON group by subtracting the average VEH/CON ΔCt value from individual ΔCt values within all experimental groups. The ΔΔCt values for each gene were converted to relative copy number (RCN) values using the following equation: RCN = 2–ΔΔCt. Normalized fold-increase for each gene was calculated by dividing individual RCN values within each experimental group by average VEH/CON RCN.

Usage notes

Microsoft Excel

Funding

National Science Foundation, Award: 1761320

National Institute of Environmental Health Sciences, Award: ES027353

National Institute on Aging, Award: AG075465

National Institute of General Medical Sciences, Award: GM142521