DOI: 10.5061/dryad.h44j0zppx
title: 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

description: 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 &lt; R-LPS/DHA ≈
 R-LPS/TPPU &lt;&lt;&lt; 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.
description: <strong>LC-MS/MS Quantitation of Plasma
 Oxylipins</strong> 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 BGB<sub>2</sub>-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.
 <strong>High-Throughput Autoantibody
 Profiling</strong> 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)
 <strong>Kidney Gene Expression</strong>
 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 (<em>Il1a,
 Il1b, Il2, Il6, Il17a, Il18</em>), chemokine (<em>Ccl2, Ccl7,
 Ccl12, Cxcl9, Cxcl10, Cxcl13</em>), inflammation/autoimmunity
 (<em>C1qa, C3, Casp1, Casp4, Icam1, Ifng, Lbp, Nfkb1, Nlrp3, Nos2,
 Pparg, Tlr4, Tlr9, Tnfa, Tnfsf13b</em>), type I interferon
 (IFN)-related (<em>Ifi44, Irf7, Isg15, Nlrc5, Oas2</em>),
 eicosanoid-related (<em>Alox15, Cyp2c44, Cyp2j6, Cyp2j9, Cyp2j11,
 Ephx1, Ephx2, Pla2g4c, Ptgs2</em>), kidney injury (<em>Ankrd1,
 Cd14, Havcr1, Tgfbr1</em>), oxidative stress-related
 (<em>Hmox, Ncf1, Nqo1, Sod2</em>), and housekeeping
 (<em>Actb</em>, <em>Gusb</em>) 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<sup>–ΔΔCt</sup>. Normalized fold-increase
 for each gene was calculated by dividing individual RCN values within each
 experimental group by average VEH/CON RCN.
description: Microsoft Excel