Black C57BL/6 WT mice (6–8 weeks old) were purchased from Charles River Inc. All animal work was conducted under UK Home Office personal and project licences, approved by the Animal Welfare Ethical Review Board (AWERB) of King’s College London and carried out in accordance with the Animals (Scientific Procedures) Act 1986 and in compliance with the ARRIVE guidelines.
Chemicals and reagents
Hydrogen peroxide (H2O2, 31642), adenosine 5′diphosphoribose (ADPR, A0752), thioglycolate (70157), lipopolysaccharide (LPS, LPS25), CXCL2 (SRP4251), complement component 5a (C5a, SRP4895A), N-(p-Amylcinnamoyl)anthranilic acid (ACA, A8486), lumefantrine (PHR2186), mefluoquine (PHR1705), hydroxychloroquine (PHR1782) and arteether (SML2592) were purchased from Sigma-Aldrich (Sigma-Aldrich Company Ltd., Dorset, UK). Pluronic F-127 (P3000MP) and Fura-2AM (F1221) were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific Life Technologies, Waltham, Massachusetts, U.S). The RAL DIFF-QUIK kit (a modified version of the May-Grünwald-Giemsa stain) (RAL555) was purchased from RAL Diagnostics, (RAL Diagnostics, Martillac, France). Deoxyartemisinin (2-deoxy-artemisinin) (20428) and artemether (11815) were purchased from Cambridge Bioscience (Cambridge UK). Artesunate (A3731), thapsigargin (T9033), desferrioxamine (BP987), L-cysteine (168149), pyrimethamine (BP1227) and dihydroartemsinin (1200520) were purchased from Merck Life Sciences (Feltham, UK). The XTT cell viability kit (9095S) was purchased from New England Biolabs (Ipswich, Massachusetts, U.S.) The natural products: artemisinin, beta-carotene, bisabolol, capsaicin, carvacrol, citral, citronellal, curcumin, D-biotin, ergosterol, eugenol, farnesene, farnesol, ferulic acid, gallic acid, geraniol, hesperidin, isoeugenol, lanosterol, lawsone, limonene, myrcene, N-acetylcysteine, neomenthol, (+) – pulegone, (-) – pulegone, quercetin, rutin-hydrate, thymol, vanillin and veratrylamine were kindly donated by Dr Suaib Luqman from CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015, Uttar Pradesh, India.
Isolation of mouse peritoneal neutrophils and macrophages
In vitro chemotaxis experiments
Mice were injected i.p. with 3% thioglycolate solution (10 μl/g) and, after 4 h (for neutrophils) or 4 d (for macrophages), were euthanised by cervical dislocation. The peritoneal-covering skin was removed, 5 ml PBS injected into the peritoneal cavity which was massaged gently for 60 s to dislodge cells. The peritoneal fluid was gently extracted by syringe and centrifuged for 10 min at 200 RCF. The supernatant was discarded and cells resuspended in DMEM + 10% FBS. These methods generated cell suspensions containing > 90% of either neutrophils or macrophages, identified through a fast-acting modified version of the May-Grünwald-Giemsa staining and subsequent cell type identification as shown in8 (neutrophils) and Supplementary Fig 3 (macrophages).
In vivo peritoneal chemotaxis experiments
Mice were injected i.p. with H2O2 or LPS and at experimental time points (see methods below), mice were euthanised by cervical dislocation. The peritoneal lavage was recovered as above, and samples of the suspensions were immediately spun down onto glass slides using a cytocentrifuge (Sigma 2-7 Cyto, ShandonTM, Germany as described below) and leukocytes (neutrophils, macrophages) identified through a fast-acting modified version of the May-Grünwald-Giemsa staining and subsequent cell type identification as shown in Morad et al. 2021. The remaining cell suspension was then centrifuged for 10 min at 200 RCF and supernatants were collected and frozen at -20oC for cyto/chemokine analysis by ELISA and cf-DNA(NET) quantification using Quant-iT™ PicoGreen kit (Thermo Fisher).
Isolation of mouse BALF neutrophils
The nostrils of mice were infused with H2O2 or LPS and at experimental time points (see lung methods below), mice were euthanised by destruction of the brain. The mice were placed in the supine position, limbs were secured and the skin around the neck was removed. Salivary glands were separated to reveal the sternal hyoid muscle and forceps used to incise the muscle around the trachea. A cotton suture was then threaded under the tracheal tissue. A needle was then used to puncture the middle of the trachea between two cartilage rings and a pre-made plastic catheter was inserted ~0.5 cm into the tracheal lumen and stabilised with the suture. A syringe, loaded with 1 ml PBS was then attached to the catheter and PBS slowly injected. The thorax was massaged gently for 60 s, before BAL fluid was aspirated. This was repeated 3 times to maximise the BAL fluid recovery.
Samples of the BAL fluid were immediately spun down onto glass slides using a cytocentrifuge (as described below) and neutrophils identified through a fast-acting modified version of the May-Grünwald-Giemsa staining and subsequent cell type identification as shown in Morad et al. 2021. The remaining cell suspensions was then centrifuged for 10 min at 200 RCF and supernatants were collected and frozen at -20°C for cyto/chemokine analysis by ELISA and cf-DNA (NET) quantification using Quant-iT™ PicoGreen kit (Thermo Fisher).
Cell identification in peritoneal and BALF extracts
Cell suspension was isolated from peritonea/lungs of WT mice as above, spun down onto glass slides using a cytocentrifuge at 400 RPM for 5 min and left to air-dry overnight. A modified version of the May-Grünwald-Giemsa staining was used to identify cell types (RAL DIFF-QUIK kit, RAL diagnostics). Slides were suspended in RAL Diff-Quick fixative solution (methanol based solution to stabilize cellular components) for 1 min, in RAL Diff-Quik solution I (Xanthene solution; a buffered solution of Eosin Y) for 1 min and in RAL Diff-Quik solution II (a buffered solution of thiazine dyes, consisting of methylene blue and Azure A) for 1 min. Nuclei were meta-chromatically stained red/purple and cytoplasm pink/yellow (see ref8 and Supplementary Fig 4).
Neutrophil and macrophage chemotaxis assays
Ibidi µ-slide chemotaxis assay chambers, precoated with collagen IV along the central migration strip, were purchased from Thistle Scientific Ltd (Uddingston, Glasgow, UK). Neutrophils or macrophages, isolated as above from peritonea of WT mice, were re-suspended within 30 min of collection in DMEM + 10% FBS at a concentration of 5 × 105 cells per ml and 6 µl was seeded along the central migration strip of an ibidi µ-slide chamber as per the manufacturer’s instructions. Slides were incubated for 1 h at 37 °C in humidified 95% air/5% CO2, to allow neutrophil/macrophage adherence to the central migration strip. DMEM (without added FBS) with and without added chemoattractant was then added to the wells on opposite sides of the central migration strip. DMEM was from Thermo Fisher Scientific Cat. No. 41966-029. For experiments in which effects of compounds were to be tested, equal concentrations were added to both DMEM + chemoattractant and DMEM wells. Slides were pre-incubated at 37 °C in 95% air/5% CO2 for 20 min to allow the generation of a gradient of chemoattractant across the 1 mm wide × 70 μm deep central cell migration strip. Live-cell time-lapse microscopy was then conducted using a 10 × lens and dark-field illumination on a Nikon Eclipse Ti-E inverted microscope equipped with the Nikon Perfect Focus System (PFS). The microscope was housed in a temperature-controlled Perspex box (Solent Scientific) at 37 °C, with slides housed in a stage-mounted block in humidified 95% air/5% CO2. A maximum of 12 individual chambers (4 individual slides, 3 chambers per slide) could be imaged per experiment by using a motorized stage. Stage movement, lens focus and image acquisition were controlled by Nikon NIS Elements software. Experiments were conducted over 2 h for neutrophils and 1 h for macrophages, with images of each assay compartment taken every 2 min. The ImageJ Fiji TrackMate plug-in was employed to track individual neutrophils/macrophages. A chemotaxis and migration plug-in, provided by ibidi, was used to calculate speed and forward migration index (FMI) data from the neutrophil/macrophage tracks.
Calcium imaging of neutrophils
Neutrophils isolated as above from the peritonea of WT mice, were re-suspended in DMEM + 10% FBS at a concentration of 5 × 105 per ml. Neutrophils were plated onto a collagen-coated 13 mm round glass coverslip and incubated at 37 °C in 95%air/5% CO2 for 1 h to allow neutrophils to adhere. Fura2-AM (5 µM in DMEM) was then added to the cells on the coverslip for 30 min at 37 °C in 95% air/5% CO2. Solutions were changed as shown in the figures and fluorescence was measured during alternating illumination at 340 nm and 380 nm (OptoScan; Cairn Research Inc, Kent, UK) every 2 s using a Nikon Eclipse Ti inverted microscope with a 40 × lens and iXon 897 EM-CCD camera controlled by WinFluor 3.2 software. F340/380 ratios were obtained using FIJI (ImageJ) and converted to calcium concentrations using the equation given by Grynkiewicz et al. with values Rmax = 2.501, Rmin = 0.103, both determined experimentally, full details in Vilar et al. and Morad et al.
For experiments when calcium signals during chemotaxis up a gradient of chemoattractant were to be recorded (as in Supplementary Video 1), 1 µl of Fura-2 AM solution (50 µg Fura-2 AM + 10 µl pluronic F-127 + 10 µl DMSO) was added to 500 µl of peritoneal neutrophil suspension and incubated for 1 h at 37 °C in 95%air/5% CO2. Fura-2 loaded cells in suspension were seeded into ibidi chambers as described above and imaged in a Nikon Ti-E microscope with a 40 × phase contrast lens. Fast-moving neutrophils located in the middle of the central cell migration strip were selected, with typically only one cell imaged per field. Calcium ratio images were obtained with alternating 340 nm and 380 nm epi-illumination supplied by stable LED light sources (Fura-LED, Cairn Research), at 500ms intervals. All images were filtered by a broad-band 510 nm filter and captured with a Photometrics Prime 95B sCMOS camera. Stage movement, focus and image acquisition were controlled by Nikon NIS Elements software. The ImageJ Fiji RatioPlus plug-in was used to generate F340/380 ratio images and a rainbow look-up table (LUT) was applied to the ratio images to indicate the level of calcium.
Transfection of HEK293 cells
Human embryonic kidney HEK293 cells were split at a confluency of 80%, resuspended in media to a concentration of 7 × 104 cells per ml and 0.5 ml was plated into a four-well plate containing 13 mm glass coverslips pre-coated with poly-d-lysine (1 mg/ml), ready for transfection the following day. Cells were transfected with 0.5 µg of a plasmid containing cDNA for SERCA1, 2 or 3 using a modified calcium-phosphate protocol, as previously described47. Cells were used for calcium imaging 2d post-transfection.
Rat SERCA1a (pMT2) was a gift from Jonathan Lytton (Addgene plasmid # 75182 ; http://n2t.net/addgene:75182 ; RRID:Addgene_75182)48. Human SERCA2a (pcDNA3.1+) was a gift from Jonathan Lytton & David MacLennan (Addgene plasmid # 75187 ; http://n2t.net/addgene:75187 ; RRID:Addgene_75187)49. Human SERCA3 (pMT2) was a gift from Jonathan Lytton & David MacLennan (Addgene plasmid # 75189; http://n2t.net/addgene:75189 ; RRID:Addgene_75189)50
Transfection of HEK293 cells with TRPM2, a kind gift from Prof Y. Mori, University of Kyoto, Japan, was carried out as described above. Manual whole-cell patch clamp recording was carried out as previously described51.
XTT cell viability assay
Peritoneal neutrophils, isolated as above, were seeded into four individual 96 well plates (2×105/well) and incubated for 1 hour at 37oC in 95% air/5% CO2 to allow adherence. Artemisinin was then added to half of the wells on all plates at a 10 µM concentration. Following incubation for: 0 hrs, 12 hrs, 24 hrs and 48 hrs, respectively, 50 µL of XTT/PMS solution was added to all wells, and plates were incubated for a further 2 hrs, before absorbance was analysed on a FLUOstar Omega microplate reader (BMG LABTECH, Buckinghamshire, UK) at 450 nm.
In vivo peritoneal H2O2 chemotaxis experiments
WT mice were injected s.c. with either sham or artemisinin/artesunate (either 28mg/kg or 6 mg/kg for both) 30 mins prior to being injected i.p. with H2O2 (10 µM in PBS, 10 µl/g body weight) or PBS alone for the control baseline group. Mice were then euthanised over 10-210 mins and peritoneal lavage was extracted and cell types identified as described above, before supernatants were analysed for cytokines/chemokines by ELISA and for NETs by cf-DNA quantification.
In vivo peritoneal LPS chemotaxis experiments
WT mice were injected s.c. with either sham or artemisinin (28mg/kg) 30 mins prior to being injected i.p. with LPS (30 ng/cavity) or PBS alone for control group. Further sham/artemisinin s.c. injections were administered at 90 and 210 mins, before mice were euthanised at 300 mins and peritoneal lavage was extracted and cell types identified as described above, before supernatants were analysed for cytokines/chemokines by ELISA and for NETs by cf-DNA quantification.
Lung BAL fluid H2O2 chemotaxis experiments
WT mice were injected s.c. with either sham or artesunate (28mg/kg or 6 mg/kg) 30 mins prior to having H2O2 (10 µM in PBS) or PBS alone for control group infused into both nostrils. Mice were then euthanised after 60 mins and BALF lavage was extracted and cell types identified as described above, before supernatants were analysed were analysed for cytokines/chemokines by ELISA and for NETs by cf-DNA quantification.
Lung BAL fluid LPS chemotaxis experiments
WT mice were injected s.c. with either sham or artesunate (28mg/kg or 6mg/kg) 30 mins prior to having LPS (300 ng in PBS) or PBS alone for control group infused into both nostrils. Further sham/artemisinin s.c. injections were administered at 90 and 210 mins, before mice were euthanised at 300 mins and BALF lavage was extracted and cell types identified as described above, before supernatants were analysed were analysed for cytokines/chemokines by ELISA and for NETs by cf-DNA quantification.
Analysis of cytokines and chemokines in peritoneal and lung fluid
At the indicated times after injection of the stimuli (H2O2 or LPS), animals were terminally anesthetized and the peritoneal lavage or BALF was collected in PBS. IL-6, IL-1β, CXCL1 and CXCL2 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) using commercial kits (DuoSet; R&D Systems) as previously described52. The results are expressed as pg/mL of each cytokine/chemokine. As a control, concentrations of these cytokines/chemokines were measured in mice injected with vehicle (PBS).
Quantification of cf-DNA (NETs) in peritoneal and lung fluid
Peritoneal lavage or BALF were collected at different time points after injection of stimuli (H2O2 or LPS) and the amount of cell free-DNA (cf-DNA) was quantified using the Quant-IT™ PicoGreen® kit (Thermo Fisher) according to the manufacturer’s instructions. The fluorescence intensity (excitation at 488 nm and emission at 525 nm wavelength), a measure of the amount of DNA, was quantified by a fluorescence reader (FlexStation 3 Microplate Reader, Molecular Devices, CA, USA) as previously described53. The results are expressed as ng/mL of cf-DNA.