Ethical approval for this study was granted by local research ethics committee at Karolinska University hospital (Dnr 02-196) and the Regional Ethical Review Board in Stockholm (Dnr 2015/2001-31/2). Full informed consent according to the Declaration of Helsinki was obtained from all patients.

Peripheral venous blood was drawn from 10 females and 6 males, aged between 27 and 81 years. Healthy controls (n = 4) and two patient groups were included: SLE (n = 9) and DM (n = 3). Patients with diagnosis SLE or DM and aged 18 or older were recruited from the Rheumatology Clinic at Karolinska University Hospital. Patients with ongoing treatment including Sendoxan (cyclophosphamide) and Benlysta (belimumab) or with kidney failure as defined by present dialysis or previous kidney transplantation were excluded. Disease activity measurements were not obtained at the time of sampling. For healthy control and patients characteristics, see Supplementary Table 1. The blood was collected in tubes containing sodium heparin (1000 U/ml).

The inhibitors (chemical probes and other high-quality tool compounds) tested here were obtained through the SGC (www.thesgc.org) and supplied by different distributers (Supplementary Table 2). Inhibitors and control drugs (Supplementary Table 2) were reconstituted at 10 mM in DMSO (D2250, Sigma-Aldrich), aliquoted in Eppendorf tubes or 96-well plates, and kept at −80°C. A fresh aliquot was used at each experiment. Diclofenac (dual COX-1/2 inhibitor) was used as positive control for inhibition of prostanoid production. Lipopolysaccharide (LPS; L6529, Sigma-Aldrich) was reconstituted in phosphate-buffered saline (PBS) (D8537, Sigma-Aldrich) to a final concentration of 0.1 mg/ml and kept at +8°C.

Inhibitors and vehicle control (DMSO) were diluted in PBS at room temperature with no direct light on. The treatments were prepared in 25 µl portions to U-shaped 96-well plate and 200 µl of freshly drawn heparin blood (< 2 h at room temperature) was added to the plate. The plate was incubated at 37°C for 30 min and then 25 µl of 0.1 mg/ml LPS in PBS was added followed by pipetting up and down 3 times (final concentration of LPS was 10 µg/ml). The tested concentration for inhibitor was 0.1 or 1 µM (Supplementary Table 1). The plate was incubated for 24 h at 37°C and then centrifuged at 3000g for 10 min at 4°C. Working on ice, 100 µl plasma was recovered to a new plate (for prostanoid profiling) and from this 20 µl was transferred to a second plate (for IL-8 quantification). The plates were sealed with aluminum foil and stored at -80°C.

Plasma samples (80–240 µl) were thawed on ice and spiked with 50 µl deuterated internal standard mix containing 17 ng 6-keto-PGF_1α-d4, 8 ng PGF_2α-d4, 12 ng PGE₂-d4, 8 ng PGD₂-d4, 8 ng thromboxane B₂ (TXB₂)-d4, and 8 ng 15-deoxy-Δ12,14PGJ₂-d4 (Cayman Chemical Company) prepared in 100% methanol. Protein precipitation was performed by addition of 800 µl 100% methanol, followed by vortexing, and centrifugation at 3000g for 10 min at 4°C. The supernatants were collected in a new plate and evaporated under vacuum for 4 h. The evaporated samples (100–200 µl) were diluted to 1 ml with 0.05% formic acid in water and then loaded onto Oasis HLB 1 cc 30 mg plate (Waters Corporation, USA) that had been pre-conditioned with 1 ml of 100% methanol and 1 ml of 0.05% formic acid in water. The plate was washed with 10% methanol, 0.05% formic acid in water and lipids were eluted with 100% methanol. The eluates were dried under vacuum overnight and stored at −20°C until reconstituted in 50 µl of 20% acetonitrile in water prior to analysis with liquid chromatography tandem mass spectrometry (LC-MS/MS).

Lipids were quantified in negative mode with multiple reaction monitoring method, using a triple quadrupole mass spectrometer (Acquity TQ detector, Waters) equipped with an Acquity H-class UPLC (Waters). Eicosanoid were purchased from Cayman Chemicals and individually optimized for based on precursor ion m/z, cone voltage, collision energy, and fragment ion m/z (Supplementary Table 3). An eicosanoid mix containing all standards of interest was used to check interference in the LC-MS/MS analysis. Lysophophatidylcholine (LPC)(14:0) and LPC(18:0) were used to set optimal analytical parameters for quantification of LPCs. Separation of lipids was performed on a 50 × 2.1-mm Acquity UPLC BEH C18 column 1.7 µm (Waters) with a 12-min stepwise linear gradient (20%–95%) at a flowrate of 0.6 ml/min with 0.05% formic acid in acetonitrile as mobile phase B and 0.05% formic acid in water as mobile phase A. Data were analyzed using MassLynx software, version 4.1, with internal standard calibration and quantification to external standard curves for prostanoids. LPCs were normalized as area-% within each injection. Only lipids with peaks intensities of signal-to-noise greater than 10 (S/N >10) were considered in our data analysis.

The whole blood assay was developed to screen for changes in multiple eicosanoids. Each eicosanoid and corresponding deuterated variant were individually optimized in the LC-MS/MS analysis. A dilution curve containing 6-keto PGF_1α-d4, PGE₂-d4, PGD₂-d4, PGF_2α-d4, TXB₂-d4, 15d-PGJ₂-d4, LTB₄-d4, LTC₄-d5, LTD₄-d5, 5-HETE-d8, 12-HETE-d8, 15-HETE-d8, and undeuterated variants of 13-HODE, RvD1, RvD2, 17-hydroxy DHA, and protectin DX was spiked into 100 µl plasma at different stages throughout the extraction. A dilution curve was spiked in water at the same step. The dilution curve ranged from 0.006 to 1.5 pmol as final amount injected on the column in the LC-MS/MS analysis. This enabled us to investigate the lower limit of quantification (LLOQ), recovery efficacy, and matrix effect for each eicosanoid. The LLOQ injected on column was considered as great (0.02–0.05 pmol), good (0.1–0.2 pmol), or poor (0.4–1.5 pmol). Eicosanoids with great LLOQ were PGE₂, PGF_2α, TXB₂, RvD1, RvD2, LTB₄, protectin DX, and 13-HODE; good LLOQ were 6-keto PGF_1α, PGD₂, 5-HETE, 15-HETE, and LTD₄; poor LLOQ were 15d-PGJ₂, 12-HETE, 17-hydroxy DHA, and LTC₄. The extraction recovery rates were 33%–125%. The response in plasma compared to 20% acetonitrile were 52%–116% due to matrix effects. The estimated LLOQ in 100 µl plasma was approximately 1 ng/ml for the best performing eicosanoids including PGE₂, TXB₂, PGF_2α, RvD1, RvD2, and protectin DX. We can conclude that the method provided similar quantitative performance in plasma for many eicosanoids.

LPS at 10 µg/ml induced PGE₂ and TXB₂ production in human whole blood, which are the two dominant eicosanoids produced under these conditions (Mazaleuskaya et al., 2016). All other eicosanoids were below the LLOQ. We chose 10 µg/ml of LPS based on the consensus in the literature for this type of assay, yielding a robust amount of PGE₂ (49 ± 4 ng/ml, n = 5 donors) and TXB₂ (24 ± 9 ng/ml, n = 5 donors). The prostanoid production was completely blocked using the dual COX-1/2 inhibitor diclofenac (10 µM). High concentration of DMSO (0.1%) slightly decreased PGE₂ production by 20% (n = 2 donors) while DMSO at 0.01% or 0.001% had no effect. The intra-assay coefficient of variation (CV, n = 20 technical replicates) was 12% and 11% for PGE₂ and TXB₂, respectively. The inter-assay CV for control material (n = 3 donors) was 20% for PGE₂ and 30% for TXB₂. This was performed on blood that was drawn, incubated, extracted, and analyzed at separate occasions. The suppression in signal due to matrix effects and/or recovery efficiency varied between donors and experiments, ranging from 10% to 70% suppression compared to signal in extracted blank (mean ± SD, n = 6 donors, PGE₂: 45% ± 25%, TXB₂: 40 ± 20%). In summary, 24-h incubation of whole blood with 10 µg/ml LPS resulted in profound induction of the COX-1/2 products PGE₂ and TXB₂ that was efficiently blocked by diclofenac at 10 µM.

IL-8 was quantified in plasma by human IL-8 (CXCL8) enzyme-linked immunosorbent assay (ELISA) development kit (3560-1H, Mabtech) according to manufacturer’s instructions.

Data are presented as mean ± SEM if not stated otherwise. Statistical analyses were performed using GraphPad Prism 6 (GraphPad Software). One-sample t-test and two-sample t-test with Bonferroni correction were used to test significant difference. Statistical significance level was set to p < 0.05.

Our screening of inhibitors suggested that selected kinase inhibitors affected prostanoid production (Figure 1). The strongest reduction in PGE₂ production was observed by MEK-1 inhibitor PD0325901 (31% ± 6%, p = 0.001, n = 4) and MEK-1/2 inhibitor trametinib (34% ± 7%, p < 0.0001, n = 15). Moderate suppression in PGE₂ concentration was found for MEK-1/2 inhibitor selumetinib (65% ± 9%, p = 0.02, n = 5), ERK-1/2 inhibitor SCH772984 (76% ± 11%, p = 0.04, n = 13), and p38 inhibitor skepinone-L (76% ± 8%, p = 0.01, n = 13). However, the tested p38 inhibitor pamapimod did not affect PGE₂ production. Two of these compounds decreased TXB₂ production, namely trametinib (63% ± 6%, p = 0.02, n = 15) and selumetinib (74% ± 7%, p = 0.02, n = 5). Diclofenac, here used as a positive control for inhibition of prostanoid production, blocked the prostanoid production while selective COX-2 inhibitor NS-398 inhibited only PGE₂ production, in agreement with previously reported data for these compounds in whole blood assay (Larsson et al., 2019). The JAK inhibitor tofacitinib increased both PGE₂ (286% ± 51%, p = 0.01, n = 6) and TXB₂ (169% ± 20%, p = 0.02, n = 6) production. The IRAK-1/4 inhibitor I slightly increased the concentrations of PGE₂ (139% ± 15%, p = 0.04, n = 7) and TXB₂ (133% ± 8%, p = 0.008, n = 7).

We chose to investigate the strongest observed effects in more detail by performing concentration–response experiments for PD0325901, trametinib, selumetinib, and tofacitinib. All three MEK inhibitors showed a concentration-dependent response on both PGE₂ and TXB₂ production while tofacitinib showed a concentration-dependent response on PGE₂ production (Figure 2).

In line with the effect on prostanoid production, reduction in IL-8 secretion was found for PD0325901 (24% ± 9%, p = 0.03, n = 3), trametinib (27% ± 5%, p < 0.0001, n = 13), and selumetinib (45% ± 10%, p = 0.03, n = 3) (Figure 3). Moderate reduction in IL-8 secretion was found for SCH772984 (62% ± 9%, p = 0.002, n = 12) and diclofenac (66% ± 8%, p = 0.003, n = 11). We could also observe that tofacitinib increased IL-8 secretion (225% ± 57%, p = 0.16, n = 3), however not with statistical significance.

We measured LPC species within our targeted LC-MS/MS analysis. LPCs are mainly generated by metabolism of membrane phosphatidylcholine by cytosolic phospholipase A₂ (Burke and Dennis, 2009). These lipids have been reported to be involved in several cellular processes; sometimes with opposing effect depending on degree of saturation, concentration, and biological context (Sevastou et al., 2013; Drzazga et al., 2014). We observed no difference in total LPC or LPC profile when whole blood was treated with LPS neither did any of the tested inhibitors alter the LPC profile (Figure 4).